Radical compounds and methods of using thereof

ABSTRACT

Disclosed are methods for performing dynamic nuclear polarization using the polarizing agents described herein. In general, the methods involve (a) providing a frozen sample in a magnetic field, wherein the frozen sample includes a polarizing agent described herein and an analyte with at least one spin half nucleus; (b) polarizing the at least one spin half nucleus of the analyte by irradiating the frozen sample with radiation having a frequency that excites electron spin transitions in the polarizing agent; (c) optionally melting the sample to produce a molten sample; and (d) detecting nuclear spin transitions in the at least one spin half nucleus of the analyte in the frozen or molten sample. In certain embodiments, the polarizing agents can be peptide-based. In these embodiments, the polarizing agents can be readily prepared by solid-phase peptide synthesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/541,334 filed Aug. 4, 2017, which is hereby incorporated herein byreference in its entirety.

BACKGROUND

There is an interest in preparing samples with high nuclear spinpolarizations with the goal of increasing signal intensities in nuclearmagnetic resonance (NMR) spectra and magnetic resonance imaging (NFRI)images Example approaches include high frequency, microwave drivendynamic nuclear polarization (DNP), para hydrogen induced polarization(PHIP), polarization of noble gases (e.g., He, Xe and, Kr), andoptically pumped nuclear polarization of semiconductors andphotosynthetic reaction centers and other proteins. Dynamic nuclearpolarization is an approach in which the large spin polarization in anelectron spin system is transferred to a nuclear spin reservoir viamicrowave irradiation of the electron paramagnetic resonance (EPR)spectrum. The electron spin system in DNP is provided by an endogenousor exogenous paramagnetic polarizing agent. A number of polarizingagents have been investigated, including monoradicals (e.g., TEMPO-basedradicals, trityl radicals, etc.) and biradicals (e.g.,bis-TEMPO-2-ethyleneglycol (BT2E), where TEMPO is2,2,6.6-tetramethylpiperidin-1-oxyl and n=2 indicates a tether of twoethylene glycol units). While some polarizing agents are known, thereremains a need in the art for improved polarizing agents and inparticular improved biradical polarizing agents

SUMMARY

Disclosed herein are compounds defined by Formula I below

wherein L represents a direct bond or a linking group; R¹ is selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, C_(M), alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycioalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene. 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, C₁₋₁₂ alkylcarbonyl,C₁₋₁₂ alkoxycarbonyl, C₁₋₁₂ alkylcarbamyl, di(C₁₋₁₂-alkyl)carbamyl,amino acid, poly(amino acid), and poly(alkylene oxide), each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(x) groups; R²is selected from the group consisting of hydrogen, hydroxy, —OR¹¹, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C₅₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁. ₄ alkylene , 5-10 membered heteroaryl-C₁₋₄alkylene, amino, C₁₋₁₂alkylamino; di(C₁₋₁₂-alkyl)amino, amino acid,poly(amino acid), and poly(alkylene oxide), each optionally substitutedwith 1, 2, 3, or 4 independently selected R^(X) groups; R³ and R⁴ areindependently selected from group consisting of C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10membered heteroaryl, 4-10 membered heterocycloalkyl,C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 memberedheterocycloalkyl-C₁₋₄alkylene, 6-10 membered aryl-C₁₋₄ alkylene, 5-10membered heteroaryl-C₁₋₄ alkylene, each optionally substituted with 1,2, 3, or 4 independently selected R^(x) groups; or R³ and R4, togetherwith the carbon atom to which they are attached, form a 3-10 memberedcycloalkyl or 4-10 membered heterocycloalkyl ring each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups; R³and R⁶ are independently selected from group consisting of C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalky, 6-10 membered aryl, 5-10membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycioatky)-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, each optionally substituted with 1, 2, 3. or 4 independentlyselected R^(X) groups, or R⁵ and R⁶, together with the carbon atom towhich they are attached, form a 3-10 membered cycloalkyl or 4-10membered lieterocycloalkyl ring each optionally substituted with 1, 2,3, or 4 independently selected R^(X) groups; R⁷ and R⁸ are independendyselected from group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocvcloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups, orR⁷ and R⁸, together with the carbon atom to which they are attached,form a 3-10 membered cycloalkyl or 4-10 membered heteroeycloalkyl ringeach optionally substituted with 1, 2, 3, or 4 independently selectedR^(X) groups, R⁹ and R¹⁰ are independently selected from groupconsisting of C₁₋₆ alkyl, C₁₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀cycloalkyl,6-10 membered aryl, 5-10 membered heteroaryl, 4-10 memberedheterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄alkylene, 4-10 memberedheterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄ alkylene, 5-10membered heteroaryl-C₁₋₄ alkylene, each optionally substituted with 1,2, 3, or 4 independently selected R^(X) groups; or R⁹ and R¹⁰ togetherwith the carbon atom to which they are attached, form a 3-10 memberedcycloalkyl or 4-10 membered heterocycloalkyl ring each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups; R¹¹is selected from the group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋ ₁₀cycloalkyl, 6-10 membered aryl. 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀ cycloalkly-C₁₋₄alkylene. 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋ ₄ alkylene , and poly(alkyleneoxide), each optionally substituted with 1, 2, 3, or 4 independentlyselected R^(X) groups, and each R^(X), when present, are eachindependently selected from OH, NO₂, CN, halo, C₁₋₆ alkyl, C₁₋₆ alkenyl,C₂₋₆ alkynyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, cyano-C₁₋₃alkyl, HO-C₁₋₃ alkyl, amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, thio.C₁₋₆ alkylthio C₁₋₆ alkylsulfinyl, C₁₋₆ alkyl sulfonyl, carbamyl, C₁₋₆alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl, carboxy, C₁₋₆ alkylcarbonyl, C₁₋₆alkoxycarbonyl, C₁₋₆ alkylcarbaylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl. C₁₋₆ alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl,aminosulfonylamino, C₁₋₆alkylaminosulfonylamino, di(C₁₋₆alkyl)aminosulfonylamino, aminocarbonylamino, C₁₋₆alkylaminocarbonylamino, and di(C₁₋₆ alkyl)aminocarbonylamino.

The compounds described herein can be used as polarizing agents forperforming dynamic nuclear polarization. Accordingly, also provided aremethods that comprise (a) providing a frozen sample in a magnetic field,wherein the frozen sample includes a polarizing agent described hereinand an analyte with at least one spin half nucleus; (b) polarizing theat least one spin half nucleus of the analyte by irradiating the frozensample with radiation having a frequency that excites electron spintransitions in the polarizing agent; (c) optionally melting the sampleto produce a molten sample, and (d) detecting nuclear spin transitionsin the at least one spin half nucleus of the analyte in the frozen ormolten sample. In certain embodiments, the methods further comprise astep of freezing a sample in a magnetic field to provide the frozensample in a magnetic field In one such embodiment, the sample is meltedprior to detection and the freezing, polarizing, melting and detectingsteps are repeated at least once.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the structures of nitroxide biradicals: AMUPOL, TOTAPOL,TOAC-TOAC (TT), TOAC-Ser-TOAC (TST), TOAC-TOAC-Ser (TTS) &acetyl-TOAC-TOAC (ATT).

FIG. 2A shows the DNP enhancement profiles of TT as a function ofconcentration: microwaves on/offsignal enhancement, εDNP, and theabsolute signal enhancement between doped and undoped samples, εAbs. TheDNP build-up time, τDNP, as a function of biradical concentration, isalso shown

FIG. 2B shows the DNP enhancement profiles of TST as a function ofconcentration: microwaves on/off signal enhancement, εDNP, and theabsolute signal enhancement between doped and undoped samples. εAbs TheDNP build-up time, τDNP, as a function of biradical concentration, isalso shown.

FIG. 2C shows the DNP enhancement profiles of TTS as a function ofconcentration microwaves on/offsignal enhancement, εDNP, and theabsolute signal enhancement between doped and undoped samples, εAbs. TheDNP build-up time, rDNP, as a function of biradical concentration, isalso shown

FIG. 2D shows the DNP enhancement profiles of ATT as a function ofconcentration microwaves on/off signal enhancement, εDNP, and theabsolute signal enhancement between doped and undoped samples, εAbs. TheDNP build-up time, τDNP, as a function of biradical concentration, isalso shown

FIG. 3 shows the time-adjusted absolute DNP enhancement profiles forAMUPOL, ATT, TT, TTS, TST, and TOTAPOL, as a function of biradicalconcentration

FIG. 4 shows 10-minute ¹H-¹³C CP experiments of the optimalconcentrations of AMUPOL, TTS, TT, ATT, TST & TOTAPOL with recycledelays set to 1.26 x τDNP.

FIG. 5A shows the positive mode electron spray ionization massspectrometry (ESI-MS) of oxidized TOAC-TOAC, TT(OH) . Expected mass:414.28 m/z. Found masses: 414.27 m/z, 415.27 m/z, 416.28 m/z. 95.6% TTpurity.

FIG. 5B shows the negative and positive mode ESI-MS of activated,reduced TOAC-TOAC, TT. Expected mass 412.27 m/z. Found masses: 411.24m/z, 412.25 m/z, 4.13 25 m/z, 413.26 m/z, 414.27 m/z. 90.4% TT purity.

FIG. 5C shows the continuous wave, X-band electron paramagneticresonance (EPR) spectrum of activated TOAC-TOAC (500 mM NH₄CH₃COO pH9.5, 50 mM TT) diluted to 5 mM TT in methanol. Microwave power = 2.0 W,sampling time = 7.0 s, time constant = 1.28 s, 4 scans.

FIG. 5D shows the continuous wave, X-band EPR spectrum of purifiedTOAC-TOAC by reverse-phase HPLC (C_(18,) H₂O/CH₃CN) and lyophilization.Dissolved in methanol Microwave power = 2.0 W. sampling time = 7.0 s,time constant = 1.28 s, 4 scans.

FIG. 6A shows negative mode ESI-MS of reduced TOAC-Ser-TOAC, TST.Expected mass: 499.30 m/z. Found masses. 498.3 m/z, 499.3 m/z, 500.3m/z. 91.3% TST purity.

FIG. 6B shows continuous wave, X-band EPR spectrum of activatedTOAC-Ser-TOAC (500 mM NH₄CH₃COO pH 9.5, 50 mM TST) diluted to 5 mM TSTin methanol. Microwave power = 2.0 W, sampling time = 7.0 s, timeconstant :1.28 s, 128 scans.

FIG. 7A shows negative mode ESI-MS of reduced TOAC-TOAC-Ser, TTS.Expected mass: 499.30 m/z. Found masses: 498.3 m/z, 499.3 m/z, 500.3m/z. 91.3% TST purity.

FIG. 7B shows continuous wave, X-band EPR spectrum of activated TOAC-TOAC-Ser (500 mM NH₄CH₃COO pH 9 5, 50 mM TST) diluted to 5 mM TTS inmethanol Microwave power = 2.0 W, sampling time = 7.0 s, time constant128 s, 128 scans

FIG. 8A shows negative mode ESI-MS of reduced Acetyl-TOAC-TOAC, ATT.Expected mass: 454.28 m/z. Found masses: 453.3 m/z, 454.3 m/z, 455.3m/z, 907.5 m/z, 908.5 m/z. 95.1% ATT purity.

FIG. 8B shows continuous wave, X-band EPR spectrum of activatedAcetyl-TOAC-TOAC in methanol. Microwave power = 2.0 W, sampling time =7.0 s, time constant = 1.28 s, 128 scans.

FIG. 9 shows the use if EPR spectroscopy to determine nitroxideconcentration. 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL,172.24 g/mol) was dissolved in 3:2 glycerol/H₂O Concentrations wereconfirmed and standardized by UV-Vis spectroscopy (λ=240 nm). EPRspectroscopy was performed on an X-band, Bruker EMX-Plus spectrometer atroom temperature EPR parameters: center field = 3350 G, microwave power= 2.0 W, sampling time = 7.0 s, time constant = 1.28 s, 4 scans. Doubleintegration of the EPR spectrum and the Q-factor of the resonator wasused to determine the electron-spin concentration.

FIG. 10 shows the EPR spectra of biradical DNP samples. 12 mM AMUPOL, 16mM TOAC-TOAC, 15 mM TOTAPOL, 17 mM TOAC-Ser-TOAC, 17 mM TOAC-TOAC-Serand 16 mM Acetyl-TOAC-TOAC biradicals were prepared in 6:3:1d⁸-glycerol/D₂O/H₂O (v/v/v) with 0.5 M ¹³C,¹⁵N-/.-proline. EPRspectroscopy was performed on a continuous wave, X-band, Bruker EMX-Plusspectrometer at room temperature. EPR parameters: center field = 3350 or3308 (different cavities) G, microwave power = 2.0 W, sampling time =7.0 s, time constant = 1.28 s, 4 scans. Signals were scaled to equalintensity for clarity.

FIG. 11A shows DNP enhancements as a function of magic angle spinning(MAS) frequency from 5 kHz to 12 kHz, where microwave-on spectra arecompared to microwave-off spectra at the same spinning speed.

FIG. 11B shows DNP enhancements as a function of temperature, withmicrowave-on spectra are compared to microwave-off spectra ofapproximately the same temperature.

FIG. 12 shows microwave power curves for TOAC-TOAC (TT), TOAC-TOAC-Ser(TTS), TOAC-Ser-TOAC (TTS), and Acetyl-TOAC-TOAC (ATT). Microwave powersare arrayed from 0.06 V (90 mA current) to 0 25 V (140 mA current) DNPenhancements from measured from 1D 1H-13C CP experiments betweenmicrowave-on spectra and a microwave-off spectrum.

FIG. 13 shows a 1D ¹H-¹³C CP experiment of ¹³C,¹⁵N-wt-huPrp23-144fibrils prepared with 12 mM AMUPOL and 12 mM TTS in 6:3:1d8-glycerol/D₂O/H₂O (v/v/v) Both DNP enhanced (microwave on) spectra andmicrowave off spectra are shown using optimal recycle delays. The numberof scans was adjusted to achieve a 10-minute experiment.

DETAILED DESCRIPTION Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control

At various places in the present specification, divalent linkingsubstituents are described. Where the structure clearly requires alinking group, the Markush variables listed for that group areunderstood to be linking groups.

The term “n-membered” where n is an integer typically describes thenumber of ring-forming atoms in a moiety where the number ofring-forming atoms is n. For example, piperidinyl is an example of a6-membered heterocycloalkyl ring, pyrazolyl is an example of a5-membered heteroaryl ring, pyridyl is an example of a 6-memberedheteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a10-membered cycloalkyl group

As used herein, the phrase ‘optionally substituted′’ means unsubstitutedor substituted. As used herein, the term “substituted” means that ahydrogen atom is removed and replaced by a substituent. It is to beunderstood that substitution at a given atom is limited by valency.

Throughout the definitions, the term “C_(n-m)” indicates a range whichincludes the endpoints, wherein n and m are integers and indicate thenumber of carbons. Examples include C₁₋₄, C₁₋₆, and the like.

As used herein, the term “C_(n-m)alkyl”, employed alone or incombination with other terms, refers to a saturated hydrocarbon groupthat may be straight-chain or branched, having n to m carbons . Examplesof alkyl moieties include, but are not limited to chemical groups suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl,sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl,n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, thealkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms,from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

As used herein, “C_(n-m) alkenyl” refers to an alkyl group having one ormore double carbon-carbon bonds and having n to m carbons. Examplealkenyl groups include, but are not limited to, ethenyl, n-propenyl,isopropenyl, n-butenyl, sec-butenyl, and the like In some embodiments,the alkenyl moiety contains 2 to 6. 2 to 4, or 2 to 3 carbon atoms.

As used herein, “C_(n-m) alkynyl” refers to an alkyl group having one ormore triple carbon-carbon bonds and having n to m carbons. Examplealkynyl groups include, but are not limited to, ethynyl, propyn-1-yl,propyn-2-yl, and the like In some embodiments, the alkynyl moietycontains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

As used herein, the term “C_(n-m) alkylene”, employed alone or incombination with other terms, refers to a divalent alkyl linking grouphaving n to m carbons. Examples of alkylene groups include, but are notlimited to, ethan-1,2-diyl, propan-1,3-diyl, propan-1,2-diyl,butan-1,4-diyl, butan-1,3-diyl, butan-1,2-diyl,2-methyl-propan-1,3-diyl, and the like. In some embodiments, thealkylene moiety contains 2 to 6, 2 to 4, 2 to 3,1 to 6, 1 to 4, or 1 to2 carbon atoms.

As used herein, the term “C_(n-m) alkoxy”, employed alone or incombination with other terms, refers to a group of formula —O—alkyl,wherein the alkyl group has n to m carbons. Example alkoxy groupsinclude methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy),tert-butoxy, and the like In some embodiments, the alkyl group has 1 to6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “C_(n-m) alkylamino” refers to a group offormula -NH(alkyl), wherein the alkyl group has n to m carbon atoms . Insome embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbonatoms.

As used herein, the term “C_(n-m)alkoxycarbonyl” refers to a group offormula —C(O)O—alkyl, wherein the alkyl group has n to m carbon atoms.In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3carbon atoms.

As used herein, the term “C_(n-m)alkylcarbonyl” refers to a group offormula —C(O)—alkyl, wherein the alkyl group has n to m carbon atoms. Insome embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbonatoms.

As used herein, the term “C_(n-m)alkylcarbonylamino” refers to a groupof formula —NHC(O)—alkyl, wherein the alkyl group has n to m carbonatoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to3 carbon atoms

As used herein, the term “C_(n-m) alkylsulfonylamino” refers to a groupof formula —NHS(O)₂—alkyl, wherein the alkyl group has n to m carbonatoms In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3carbon atoms.

As used herein, the term “aminosulfonyl” refers to a group of formula—S(O)₂NH₂.

As used herein, the term “C_(n-m)alkylaminosulfonyl” refers to a groupof formula -S(O)₂NH(alkyl), wherein the alkyl group has n to m carbonatoms . In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to3 carbon atoms.

As used herein, the term “di(C_(n-m) alkyl)aminosulfonyl” refers to agroup of formula -S(O)₂N(alkyl)₂, wherein each alkyl group independentlyhas n to m carbon atoms. In some embodiments, each alkyl group has,independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms

As used herein, the term “aminosulfonylamino” refers to a group offormula -NHS(O)₂NH_(2.)

As used herein, the term “C_(n-m)alkylaminosulfonylamino” refers to agroup of formula -NHS(O)₂NH(alkyl), wherein the alkyl group has n to mcarbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4,or 1 to 3 carbon atoms.

As used herein, the term “di(C_(n-m) alkyl)aminosulfonylamino” refers toa group of formula -NHS(O)₂N(alkyl)₂, wherein each alkyl groupindependently has n to m carbon atoms . In some embodiments, each alkylgroup has, independently, 1 to 6. 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “aminocarbonylamino”, employed alone or incombination with other terms, refers to a group of formula —NHC(O)NH₂

As used herein, the term “C_(n-m) alkylaminocarbonylamino” refers to agroup of formula -NHC(O)NH(alkyl), wherein the alkyl group has n to mcarbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4,or 1 to 3 carbon atoms.

As used herein, the term “di(C_(n-m) alkyl)aminocarbonylamino” refers toa group of formula -NHC(O)N(alkyl)₂, wherein each alkyl groupindependently has n to m carbon atoms. In some embodiments, each alkylgroup has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms

As used herein, the term “C_(n•m)alkylcarbamyl”refers to a group offormula —C(O)—N(alkyl), wherein the alkyl group has n to m carbon atoms.In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3carbon atoms.

As used herein, the term “thio” refers to a group of formula —SH.

As used herein, the term “C_(n•m) alkylsulfinyl” refers to a group offormula —S(O)—alkyl, wherein the alkyl group has n to m carbon atoms .In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3carbon atoms.

As used herein, the term “(C_(n-m)alkylsulfony”refers to a group offormula —S(O)₂—alkyl, wherein the alkyl group has n to m carbon atoms,In some embodiments, the alkyl group has 1 to 6, 1 to 4. or 1 to 3carbon atoms

As used herein, the term “amino” refers to a group of formula --NH_(2.)

As used herein, the term “aryl,” employed alone or in combination withother terms, refers to an aromatic hydrocarbon group, which may bemonocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings). The term“C_(n•m) aryl” refers to an aryl group having from n to m ring carbonatoms. Aryl groups include, e g., phenyl, naphthyl, anthracenyl,phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, arylgroups have from 6 to about 20 carbon atoms, from 6 to about 15 carbonatoms, or from 6 to about 10 carbon atoms. In some embodiments, the arylgroup is a substituted or unsubstituted phenyl.

As used herein, the term “carbamy” to a group of formula --C(0)NH₂

As used herein, the term “carbonyl”, employed alone or in combinationwith other terms, refers to a —C( ═O)— group, which may also be writtenas C(O).

As used herein, the term “di(Cn•n-alkyl)amino”refers to a group offormula -N(alkyl)2, wherein the two alkyl groups each has,independently, n to m carbon atoms. In some embodiments, each alkylgroup independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “di(C_(n•m)-alkyl)carbamyl” refers to a groupof formula ---C(O)N(alkyl)₂, wherein the two alkyl groups each has,independently, n to m carbon atoms In some embodiments, each alkyl groupindependently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “halo” refers to F, Cl, Br, or L. In someembodiments, a halo is F. Cl, or Br. In some embodiments, a halo is F orCl

As used herein, “C_(n)-_(m) haloalkoxy” refers to a group of formula—O—haloalkyl having n to m carbon atoms. An example haloalkoxy group isOCF₃. In some embodiments, the haloalkoxy group is fluorinated only Insome embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbonatoms.

As used herein, the term “C_(n•m)haloalkyl”, employed alone or incombination with other terms, refers to an alkyl group having from onehalogen atom to 2 s+1 halogen atoms which may be the same or different,where “s” is the number of carbon atoms in the alkyl group, wherein thealkyl group has n to m carbon atoms. In some embodiments, the haloalkylgroup is fluorinated only. In some embodiments, the alkyl group has 1 to6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, “cycloalkyl” refers to non-aromatic cyclic hydrocarbonsincluding cyclized alkyl and/or alkenyl groups Cycloalkyl groups caninclude mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groupsand spirocycles Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, or 10ring-forming carbons (C₃₋₁₀). Ring-forming carbon atoms of a cycloalkylgroup can be optionally substituted by oxo or sulfido (e g., C(O) orC(S)). Cycloalkyl groups also include cycloalkylidenes. Examplecycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl,cycloheptatrienyl, norbornyl, norpinyl, norcamyl, and the like. In someembodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclopentyl, or adamantyl. In some embodiments, thecycloalkyl has 6-10 ring-forming carbon atoms. In some embodiments,cycloalkyl is adamantyl. Also included in the definition of cycloalkylare moieties that have one or more aromatic rings fused (i.e., having abond in common with) to the cycloalkyl ring, for example, benzo orthienyl derivatives of cyclopentane, cyclohexane, and the like. Acycloalkyl group containing a fused aromatic ring can be attachedthrough any ring-forming atom including a ring-forming atom of the fusedaromatic ring.

As used herein, “heteroaryl” refers to a monocyclic or polycyclicaromatic heterocycle having at least one heteroatom ring member selectedfrom sulfur, oxygen, and nitrogen. In some embodiments, the heteroarylring has 1, 2, 3, or 4 heteroatom ring members independently selectedfrom nitrogen, sulfur and oxygen. In some embodiments, any ring-formingN in a heteroaryl moiety can be an N-oxide In some embodiments, theheteroaryl has 5-10 ring atoms and 1, 2, 3 or 4 heteroatom ring membersindependently selected from nitrogen, sulfur and oxygen. In someembodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatomring members independently selected from nitrogen, sulfur and oxygen Insome embodiments, the heteroaryl is a five-membered or six-memberetedheteroaryl ring. A five-membered heteroaryl ring is a heteroaryl with aring having five ring atoms wherein one or more (e.g., 1, 2, or 3) ringatoms are independently selected from N, O, and S. Exemplaryfive-membered ring heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl,thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl,1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2.3-oxadiazolyl,1,2,4-triazolyl, 1,2.4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl,1,3,4-thiadiazolyl, and1,3,4-oxadiazolyl. A six-membered heteroaryl ringis a heteroaryl with a ring having six ring atoms wherein one or more(e.g., 1, 2, or 3) ring atoms are independently selected from N, O, andS. Exemplary six-membered ring heteroaryls are pyridyl, pyrazinyl,pyrimidinyl, triazinyl and pyridazinyl.

As used herein, “heterocycloalkyl” refers to non-aromatic monocyclic orpolycyclic heterocycles having one or more ring-forming heteroatomsselected from O, N, or S. Included in heterocycloalkyl are monocyclic4-. 5-, 6-, and 7-membered heterocycloalkyl groups Heterocycloalkylgroups can also include spirocycles. Example heterocycloalkyl groupsinclude pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl,tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino,piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl,pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl,oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, andthe like. Ring-forming carbon atoms and heteroatoms of aheterocycloalkyl group can be optionally substituted by oxo or sulfido(e.g., C(O), S(O), C(S), or S(O)₂, etc.). The heterocycloalkyl group canbe attached through a ring-forming carbon atom or a ring-formingheteroatom. In some embodiments, the heterocycloalkyl group contains 0to 3 double bonds In some embodiments, the heterocycloalkyl groupcontains 0 to 2 double bonds Also included in the definition ofheterocycloalkyl are moieties that have one or more aromatic rings fused(i.e., having a bond in common with) to the cycloalkyl ring, forexample, benzo or thienyl derivatives of piperidine, morpholine,azepine, etc. A heterocycloalkyl group containing a fused aromatic ringcan be attached through any ring-forming atom including a ring-formingatom of the fused aromatic ring. In some embodiments, theheterocycloalkyl has 4-10, 4-7 or 4-6 ring atoms with 1 or 2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur and having oneor more oxidized ring members.

At certain places, the definitions or embodiments refer to specificrings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwiseindicated, these rings can be attached to any ring member provided thatthe valency of the atom is not exceeded. For example, an azetidine ringmay be attached at any position of the ring, whereas a pyridin-3-yl ringis attached at the 3-position.

“Linker,” “Linking Moiety,” or “Linking Group”, as used herein, refer toa bivalent group or moiety which connects the two radical moieties inthe compounds described herein. The linker can be composed of anyassembly of atoms, including oligomeric and polymeric chains; however,the total number of atoms in the spacer group is preferably between 3and 200 atoms, more preferably between 3 and 150 atoms, more preferablybetween 3 and 100 atoms, more preferably between 3 and 50 atoms, mostpreferably between 3 and 20 atoms. The linker can serve to modify thesolubility of the compounds described herein. In some embodiments, thelinker is hydrophilic. In some embodiments, the linker is hydrophobic.In some embodiments, the linker can be an alkyl group, an alkylarylgroup, an oligo- or polyalkylene oxide chain (e.g., an oligo- orpolyethylene glycol chain), or an oligo- or poly(amino acid) chain.

The term “amino acid,” as used herein, refers to both natural andnon-natural amino acids, and analogs and derivatives thereof. The term“non-natural amino acid” refers to an organic compound that is acongener of a natural amino acid in that it has a structure similar to anatural amino acid so that it mimics the structure and reactivity of anatural amino acid. The non-natural amino acid can be a modified aminoacid, and/or amino acid analog, that is not one of the 20 commonnaturally occurring amino acids or the rare natural amino acidsselenocysteine or pyrrolysine. Examples of suitable amino acids include,but are not limited to, alanine, allosoleucine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, napthylalanine, phenylalanine,proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine,valine, a derivative, or combinations thereof These are listed in thetable 1 along with their abbreviations used herein.

Amino Acid Abbreviations* L-amino acid Abbreviations* D-amino acidAlanine Ala (A) ala (a) Allosoleucine Alle aile Arginine Arg (R) arg (r)Asparagine Asn (N) asn (n) aspartic acid Asp (D) asp (d) Cysteine Cys(C) cys (c) Cyclohexylalanine Cha cha 2,3-diaminopropionic acid Dap dap4-fluorophenylalanine Fpa (Σ) pfa glutamic acid Glu (E) glu (e)Glutamine Gln(Q) gln (q) Glycine Gly (G) gly (g) Histidine His (H) his(h) Homoproline (aka pipecolic acid) Pip (Θ) pip (Θ) Isoleucine Ile (I)ile (i) Leucine Leu (L) leu (l) Lysine Lys (K) lys (k) methionine Met(M) met (m) napthylalanine Nal (Φ) nal (Φ) norleucine Nle (Ω) nlephenylalanine Phe (F) phe (F) phenylglycine Phg (Ψ) phg4-(phosphonodifluoromethyl)phenylalanine F₂Pmp (Λ) f₂pmp proline Pro (P)pro (p) sarcosine Sar sar selenocysteine Sec (U) sec (u) serine Ser (S)ser (s) threonine Thr (T) thr (y) tyrosine Tyr (Y) tyr (y) tryptophanTrp (W) trp (w) 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acidTOAC (TOAC) toac (toac) Valine Val (V) val (v) 2,3-diaminopropionic acidDap dap * single letter abbreviations: when shown in capital lettersherein it indicates the L-amino acid form, when shown in lower caseherein it indicates the D-amino acid form

As discussed above, non-natural amino acids and D-amino acids can beused herein. In some cases, amino acids can be coupled by a peptidebond. Each amino acid can be coupled to an adjacent amino acid at theamino group, the carboxylate group, or the side chain

The term “poly(amino acid),” as used herein, refers to a moietycomprising a plurality of amino acids coupled by peptide bondsPoly(amino acid) groups can comprise from 2 to 500, from 2 to 250, from2 to 100, from 2 to 50, from 2 to 25, from 2 to 20, from 2 to 15, from 2to 10, from 2 to 5, from 2 to 4, or from 2 to 3 amino acid residues. Insome embodiments, the poly(amino acid) group can be a dipeptide In someembodiments, the poly(amino acid) group can be a tripeptide. In someembodiments, the poly(amino acid) group can be a tetrapeptide. In someembodiments, the poly(amino acid) group can comprise a protein Incertain embodiments, the protein can be an analyte of interest.

The term “direct bond” or “bond” refers to a single, double or triplebond between two groups. In certain embodiments, a “direct bond” refersto a single bond between two groups

The term “compound” as used herein is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted Compounds herein identified by name or structure asone particular tautomeric form are intended to include other tautomericforms unless otherwise specified.

Compounds provided herein also include tautomeric forms . Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton. Tautomericforms include prototropic tautomers which are isomeric protonationstates having the same empirical formula and total charge. Exampleprototropic tautomers include ketone - enol pairs, amide - imidic acidpairs, lactam - lactim pairs, enamine imine pairs, and annular formswhere a proton can occupy two or more positions of a heterocyclicsystem, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H-and 2H- isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution

In some embodiments, the compounds described herein can contain one ormore asymmetric centers and thus occur as racemates and racemicmixtures, enantiomerically enriched mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures (e.g., including(R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (+)(dextrorotatory) forms. (-) (levorotatory) forms, the racemic mixturesthereof, and other mixtures thereof). Additional asymmetric carbon atomscan be present in a substituent, such as an alkyl group. All suchisomeric forms, as well as mixtures thereof, of these compounds areexpressly included in the present description The compounds describedherein can also or further contain linkages wherein bond rotation isrestricted about that particular linkage, e g. restriction resultingfrom the presence of a ring or double bond (e.g., carbon-carbon bonds,carbon-nitrogen bonds such as amide bonds). Accordingly, all cis/transand E/Z isomers and rotational isomers are expressly included in thepresent description. Unless otherwise mentioned or indicated, thechemical designation of a compound encompasses the mixture of allpossible stereochemically isomeric forms of that compound.

Optical isomers can be obtained in pure form by standard proceduresknown to those skilled in the art, and include, but are not limited to,diastereomeric salt formation, kinetic resolution, and asymmetricsynthesis See, for example, Jacques, et al , Enantiomers, Racemates andResolutions (Wiley Interscience. New York, 1981); Wilen, S.H., et al.,Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of CarbonCompounds (McGraw-Hill, NY. 1962); Wilen, S.H. Tables of ResolvingAgents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of NotreDame Press. Notre Dame, IN 1972), each of which is incorporated hereinby reference in their entireties. It is also understood that thecompounds described herein include all possible regioisomers, andmixtures thereof, which can be obtained in pure form by standardseparation procedures known to those skilled in the art, and include,but are not limited to, column chromatography, thin-layerchromatography, and high-performance liquid chromatography.

Unless specifically defined, compounds provided herein can also includeall isotopes of atoms occurring in the intermediates or final compounds.Isotopes include those atoms having the same atomic number but differentmass numbers. Unless otherwise stated, when an atom is designated as anisotope or radioisotope (e.g., deuterium, [¹¹C], [¹⁸F]), the atom isunderstood to comprise the isotope or radioisotope in an amount at leastgreater than the natural abundance of the isotope or radioisotope. Forexample, when an atom is designated as “D” or “deuterium”, the positionis understood to have deuterium at an abundance that is at least 3000times greater than the natural abundance of deuterium, which is 0.015%(i.e., at least 45% incorporation of deuterium)

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.hydrates and solvates) or can be isolated.

In some embodiments, preparation of compounds can involve the additionof acids or bases to affect, for example, catalysis of a desiredreaction or formation of salt forms such as acid addition salts

Example acids can be inorganic or organic acids and include, but are notlimited to, strong and weak acids. Some example acids includehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,p-toluenesulfonic acid, 4-nitirobenzoic acid, methanesulfonic acid,benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weakacids include, but are not limited to acetic acid, propionic acid,butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoicacid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid

Example bases include lithium hydroxide, sodium hydroxide, potassiumhydroxide, lithium carbonate, sodium carbonate, potassium carbonate, andsodium bicarbonate Some example strong bases include, but are notlimited to, hydroxide, alkoxides, metal amides, metal hydrides, metaldialkylamides and arylamines, wherein; alkoxides include lithium, sodiumand potassium salts of methyl, ethyl and t-butyl oxides; metal amidesinclude sodium amide, potassium amide and lithium amide; metal hydridesinclude sodium hydride, potassium hydride and lithium hydride, and metaldialkylamides include lithium, sodium, and potassium salts of methyl,ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, trimethylsilyl andcyclohexyl substituted amides.

In some embodiments, the compounds provided herein, or salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compounds providedherein. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compounds provided herein, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

The expressions, “ambient temperature” and “room temperature” or “rt” asused herein, are understood in the art, and refer generally to atemperature, e.g. a reaction temperature, that is about the temperatureof the room in which the reaction is carried out, for example, atemperature from about 20° C. to about 30° C.

Compounds

The sensitivity in solid-state NMR (ssNMR) experiments can be enhancedby two to three orders of magnitude by dynamically polarizing thenuclear spin system prior to recording the NMR spectrum This enhancementcan be transferred into the liquid-state (e.g., liquid-state NMR or MRI)by melting the solid sample after polarization. For MRI applications, atleast a portion of the molten sample that includes a polarized analyteis administered into the subject being imaged prior to imaging

The DNP procedure involves microwave irradiation of the electronparamagnetic resonance (EPR) spectrum of either an endogenous orexogenous paramagnetic species present in a sample, and results in thetransfer of the greater spin polarization of the electrons to the nucleiof surrounding molecules. While the methods described herein are notlimited to any specific magnetic field and the DNP procedure could beperformed at low magnetic fields, the performance of dynamic nuclearpolarization (DNP) experiments at the high magnetic fields used incontemporary NMR experiments (e.g., 5-20 T) is affected by the followingthree factors.

First, a high frequency (140-600 GHz), high power (‘10 watts) microwavesource is typically used to drive the continuous-wave (CW) DNPtransitions associated with the second order electron-nuclear dipolarinteractions (though other sources, such as pulsed and chirped microwaveirradiation can also be used). To date this has been achieved byutilizing gyrotrons since they operate in the requisite frequency rangeand produce suitable microwave powers.

Second, the relaxation times of the spin systems in the experimentdictate that it be optimally performed at low temperatures (usually ≦90K). When obtaining high resolution ssNMR spectra of solids, magic-anglespinning (MAS) is preferably incorporated into the experiment Thus,multiple resonance—i.e., ¹H, ¹³C., ¹⁵N and e⁻--low temperature MASprobes may be required for optimal execution of certain DNP experiments.

The third factor is the nature of the paramagnetic polarizing agent.Preferably, the polarizing agent should: (a) be compatible with thepolarization mechanism that yields the optimal signal enhancement,namely the three-spin thermal mixing (TM)or cross effect (CE), (b) beuseful in polarizing a large array of analytes ranging from smallmolecules to proteins, (c) produce large signal enhancements at areduced concentration of paramagnetic species, and (d) be soluble inaqueous media.

The compounds described herein can satisfy one or more of the criteriaabove. In some embodiments, the compounds described herein can satisfyat least the first three criteria In certain embodiments, can satisfythe first three criteria as well as the fourth requirement (ie.,solubility in aqueous media).

Provided herein are compounds defined by Formula I below

wherein L represents a direct bond or a linking group; R¹ is selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl. 5-10 membered heteroaryl,4-10 numbered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, (C₁₋₁₂ alkylcarbonyl,C₁₋₁₂ alkoxycarbonyl, C₁₋₁₂ alkylcarbamyl, di(C₁₋₁₂-alkyl)carbamyl,amino acid, poly(amino acid), and poly(alkylene oxide), each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups; R²is selected from the group consisting of hydrogen, hydroxy, —OR¹¹, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁-₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, amino, C₁₋₁₂ alkylamino, di(C₁₋₁₂-alkyl)amino, amino acid,poly(amino acid), and poly(alkylene oxide), each optionally substitutedwith 1, 2, 3, or 4 independently selected R^(X) groups; R³ and R⁴ areindependently selected from group consisting of C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, each optionally substituted with 1, 2, 3. or 4 independentlyselected R^(X) groups; or R³ and R⁴, together with the carbon atom towhich they are attached, form a 3-10 membered cycloalkyl or 4-10membered heterocycloalkyl ring each optionally substituted with 1, 2, 3,or 4 independently selected R^(X) groups; R⁵ and R⁶ are independentlyselected from group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, each optionallysubstituted with 1. 2. 3, or 4 independently selected R^(x) groups; orR⁵ and R⁶ together with the carbon atom to which they are attached, forma 3-10 membered cycloalkyl or 4-10 membered heterocycloalkyl ring eachoptionally substituted with 1, 2, 3, or 4 independently selected R^(X)groups; R⁷ and R⁸ are independently selected from group consisting ofC₁₋₆ alkyl, C₂₋₆ alkenyl, C₂-₆ alkynyl, C₃₋₁₀ cycloalkyl. 6-10 memberedaryl, 5-10 membered heteroaryl. 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, each optionally substituted with 1, 2, 3. or 4 independentlyselected R^(x) groups; or R⁷ and R⁸, together with the carbon atom towhich they are attached, form a 3-10 membered cycloalkyl or 4-10membered heterocycloalkyl ring each optionally substituted with 1, 2, 3,or 4 independently selected R^(X) groups; R⁹ and R¹⁰ are independentlyselected from group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups; orR⁹ and R¹⁰, together with the carbon atom to which they are attached,form a 3-10 membered cycloalkyl or 4-10 membered heterocycloalkyl ringeach optionally substituted with 1, 2, 3, or 4 independently selectedR^(X) groups; R¹¹ is selected from the group consisting of C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, cycloalkyl, 6-10 membered aryl, 5-10membered heteroaryl, 4-10 membered heterocycloalkyl. C₃₋₁₀cycloalkyl-C₁₋₄alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋ ₄alkylene, and poly(alkylene oxide), each optionally substituted with 1,2, 3, or 4 independently selected R^(X) groups; and each R^(X), whenpresent, are each independently selected from OH, NO₂, CN, halo, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl. C₁₋₄ haloalkyl, C₁₋₆ alkoxy, C₁₋₆haloalkoxy, cyano-C₁₋₃ alkyl, HO-C₁₋₃ alkyl, amino, C₁₋₆ alkylamino,di(C₁₋₆) alkyl)amino, thio, C₁₋₁₆ alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆alkylsulfonyl, carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl,carboxy. C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl, C₁₋₆alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl, aminosulfonylamino,C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆ alkyl)aminosulfonylamino,aminocarbonylamino, C₁₋₆ alkylaminocarbonylamino, and di(C₁₋₆alkyl)aminocarbonylamino.

In some embodiments, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each C₁₋₆alkyl (C₁₋₄ alkyl).

In certain embodiments, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are eachethyl.

In certain embodiments, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are eachmethyl. In these embodiments, the compound is defined by Formula IA

wherein L represents a direct bond or a linking group; R¹ is selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃-₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, C₁₋₁₂ alkylcarbonyl,C₁₋₁₂ alkoxycarbonyl, C₁₋₁₂ alkylcarbamyl, di(C₁₋₁₂-alkyl)carbamyl,amino acid, poly(amino acid), and poly(alkylene oxide), each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups, R²is selected from the group consisting of hydrogen, hydroxy, —OR¹¹, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁ ₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene,amino, (C₁₋₁₂ alkylamino; di(C₁₋₁₂-alkyl)amino, amino acid, poly(aminoacid), and poly(alkylene oxide), each optionally substituted with 1, 2,3, or 4 independently selected R^(X) groups; R¹¹ is selected from thegroup consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl. 4-10 memberedheterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene. 4-10 memberedheterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄ alkylene, 5-10membered heteroaryl-C₁-₄ alkylene, and poly(alkylene oxide), eachoptionally substituted with 1, 2, 3, or 4 independently selected R^(X)groups; and each R^(X), when present, are each independently selectedfrom OH, NO₂, CN, halo, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl. C₁₋₄haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, cyano-C₁₋₃ alkyl, HO-C₁₋₃alkyl, amino, (C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, thio, C₁₋₆alkylthio. C₁₋₆ alkylsulfinyl, C₁₋₆ alkylsulfonyl, carbamyl, C₁₋₆alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl, carboxy. C₁₋₆ alkylcarbonyl, C₁₋₆alkoxycarbonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆alkyl)aminosulfonylamino, aminocarbonylamino, C₁₋₆alkylaminocarbonylamino, and di(C₁₋₆ alkyl)aminocarbonylamino.

In other embodiments, the compound can be defined by Formula IB

wherein L represents a direct bond or a linking group; R¹ is selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, (C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, C₁₋₁₂ alkylcarbonyl,C₁₋₁₂ alkoxycarbonyl, C₁₋₁₂ alkylcarbamyl, di(C₁₋₁₂-alkyl)carbamyl,amino acid, poly(amino acid), and poiy(alkylene oxide), each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups; R²is selected from the group consisting of hydrogen, hydroxy, —OR¹¹, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryi, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋ ₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, amino, C₁₋₁₂ alkylamino; di(C₁₋₁₂-alkyl)amino, amino acid,poly(amino acid), and poly(alkylene oxide), each optionally substitutedwith 1, 2, 3, or 4 independently selected R^(X) groups, R¹¹ is selectedfrom the group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10 memberedheterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄ alkylene, 5-10membered heteroaryl-C₁₋ ₄ alkylene, and poly(alkylene oxide), eachoptionally substituted with 1, 2, 3, or 4 independently selected R^(X)groups; and each R^(X), when present, are each independently selectedfrom OH, NO₂, CN, halo, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₄haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, cyano-C₁₋₃ alkyl, HO-C₁₋₃alkyl, amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, thio, C₁₋₆alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆ alkylsulfonyl, carbamyl, C₁₋₆alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl, carboxy, C₁₋₆ alkylcarbonyl, C₁₋₆alkoxycarbonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆alkyl)aminosulfonylamino, aminocarbonylamino, C₁₋₆alkylaminocarbonylamino, and di(C₁₋₆ alkyl)aminocarbonylamino .

In other embodiments, the compound is defined by Formula IC

wherein L represents a direct bond or a linking group; R¹ is selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, C₁₋₁₂ alkylcarbonyl,C₁₋₁₂ alkoxycarbonyl, C₁₋₁₂ alkylcarbamyl, di(C₁₋₁₂-alkyl)carbamyl,amino acid, poly(amino acid), and poly(alkylene oxide), each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups, R²is selected from the group consisting of hydrogen, hydroxy, —OR¹¹, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁-₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, amino, C₁₋₁₂ alkylamino; di(C₁₋₁₂-alkyl)amino, amino acid,poly(amino acid), and poly(alkylene oxide), each optionally substitutedwith 1, 2, 3, or 4 independently selected R^(X) groups; R¹¹ is selectedfrom the group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10 memberedheterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄ alkylene, 5-10membered heteroaryl-C₁₋ ₄ alkylene, and poly(alkylene oxide), eachoptionally substituted with 1, 2, 3, or 4 independently selected R^(X)groups, R¹² is selected from the group consisting of OH, NO₂, CN, halo,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl. C₁₋₄ haloalkyl, C₁₋₆ alkoxy,C₁₋₆ haloalkoxy, cyano-C₁₋₃ alkyl, HO-C₁₋₃ alkyl, amino, C₁₋₆alkylamino, di(C₁₋₆ alkyl)amino, thio, C₁₋₆ alkylthio, C₁₋₆alkylsulfinyl, C₁₋₆ alkylsulfonyl, carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₆alkyl)carbamyl, carboxy, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl, C₁₋₆alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl, aminosulfonylamino,C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆ alkyl)aminosulfonylamino,aminocarbonylamino, C₁₋₆ alkylaminocarbonylamino, di(C₁₋₆alkyl)aminocarbonylamino, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, and poly(alkylene oxide); and each R^(X), when present, areeach independently selected from OH, NO₂, CN, halo, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₁₋₄ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,cyano-C₁₋₃ alkyl, HO-C₁₋₃ alkyl, amino, C₁₋₆ alkylamino, di(C₁₋₆alkyl)amino, thio, C₁₋₆ alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆alkylsulfonyl, carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl,carboxy, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl, C₁₋₆alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl, aminosulfonylamino,C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆ alkyl)aminosulfonylamino,aminocarbonylamino, C₁₋₆ alkylaminocarbonylamino, and di(C₁₋₆alkyl)aminocarbonylamino.

In some embodiments. L represents a direct bond. For example, thecompounds of Formula I can be represented by the formula below.

In other embodiments, L represents a linking group . In someembodiments, the linking group can comprise one or more amino acidresidues. In certain embodiments, the linking group can consist of oneor more amino acid residues (i.e., the compound can be a peptide-basedbiradical). For example, L can be defined by the structure below

wherein for each occurrence in L, R¹⁴ is H and R¹³ is selected from oneof the following

or R¹³ and R¹⁴, together with the atoms to which they are attached, forma five-membered heterocycle defined by the structure below

, and m is an integer selected from 1, 2, 3, 4, 5, and 6. In certainembodiments, m is 1. In other embodiments, m is 2. In some of theseembodiments, L can comprise one or more amino acid residues selectedfrom the group consisting of a serine residue, a threonine residue, anasparagine residue, a glutamine residue, an aspartic acid residue, acysteine residue, a glutamic acid residue, and any combination thereof.

In some embodiments, L can comprise one or more unnatural amino acids.For example, L can comprise one or more2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid (TOAC) residues .In these embodiments, the compound can comprise, for example, three,four, five, six. seven eight, or more radicals.

In some embodiments, R¹ can comprise hydrogen. In other embodiments, R¹can comprise a C₁₋₁₂ alkylcarbonyl or C₁₋₁₂ alkoxycarbonyl group. Forexample, R¹ can comprise an acetyl group. In some embodiments, R²comprises an amino acid residue. In certain embodiments, the amino acidresidue is selected from the group consisting of a serine residue, athreonine residue, an asparagine residue, a glutamine residue, anaspartic acid residue, and a glutamic acid residue . In someembodiments, R¹ comprises a cysteine residue. In these cases, thecysteine residue can be used to covalently attach the polarizing agentto an analyte (e.g., a protein by way of a disulfide bond). In someembodiments, R¹ can comprise one or more unnatural amino acids. Forexample, R¹ can comprise one or more2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid (TOAC) residues. Inthese embodiments, the compound can comprise, for example, three, four,five, six, seven eight, or more radicals In some embodiments, R¹ cancomprise a poly(amino acid) sequence. The poly(amino acid) sequence can,for example, serves as a biomolecular recognition motif for an analyteof interest.

In some embodiments, R² can comprise a hydroxy group In otherembodiments. R² can comprise OR¹¹, where R¹¹ is selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl.6-10 membered aryl, 5-10 membered heteroaryl. 4-10 memberedheterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10 memberedheterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄ alkylene, 5-10membered heteroaryl-C₁₋₄alkylene, and poly(alkylene oxide), eachoptionally substituted with 1, 2, 3, or 4 independently selectedR^(x)groups In certain embodiments, R² can comprise OR¹¹ where R¹¹ is aC₁₋₆ alkyl group optionally substituted with 1, 2, 3, or 4 independentlyselected R^(x) groups. In certain embodiments, R² can comprise OR¹¹where R¹¹ is a C₁₋₆ a poly(alkylene oxide) group.

In some embodiments, R² comprises an amino acid residue. In certainembodiments, the amino acid residue is selected from the groupconsisting of a serine residue, a threonine residue, an asparagineresidue, a glutamine residue, an aspartic acid residue, and a glutamicacid residue. In some embodiments, R² comprises a cysteine residue. Inthese cases, the cysteine residue can be used to covalently attach thepolarizing agent to an analyte (e.g., a protein by way of a disulfidebond). In some embodiments, R² can comprise one or more unnatural aminoacids For example, R² can comprise one or more2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid (TOAC) residues. Inthese embodiments, the compound can comprise, for example, three, four,five, six, seven eight, or more radicals In some embodiments, R² cancomprise a poly(amino acid) sequence. The poly(amino acid) sequence can,for example, serves as a biomolecular recognition motif for an analyteof interest

In certain embodiments, the compound can be one of the following:

In some embodiments, the compounds described herein can be deuterated atone or more positions For example, in some embodiments of Formula I, oneor more of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ can be deuterated (e.g,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ can all be -CD₃). In otherembodiments, deuterated moieties can be encorporated at R¹, R², L, or atother positions (e.g., at the meta positions of the TEMPO ring).

Methods of Use

In general, the methods involve (a) providing a frozen sample in amagnetic field, wherein the frozen sample includes a polarizing agentand an analyte with at least one spin half nucleus; (b) polarizing theat least one spin half nucleus of the analyte by irradiating the frozensample with radiation having a frequency that excites electron spintransitions in the polarizing agent; (c) optionally melting the sampleto produce a molten sample; and (d) detecting nuclear spin transitionsin the at least one spin half nucleus of the analyte in the frozen ormolten sample. The polarizing agent can be a compound (e.g., abiradical) described herein. In certain embodiments, the methods furthercomprise a step of freezing a sample in a magnetic field to provide thefrozen sample in a magnetic field In one such embodiment, the sample ismelted prior to detection and the freezing, polarizing, melting anddetecting steps are repeated at least once

In certain embodiments, the analyte is a molecule (e.g., a protein) thatis being studied by solid- or liquid-state NMR In other embodiments, theanalyte is an imaging agent that is being used for MRI In suchembodiments, the step of detecting is performed after at least a portionof the molten sample which comprises the polarized imaging agent hasbeen administered to the subject being imaged. In general, the frozensample may include any solvent; however, in certain embodiments, thefrozen sample includes an amount of water, e.g., at least 50%, 60%, 70%,80%, 90%, 95%, 99% or 100% by volume of water

In one embodiment, the methods do not include a step of melting thesample to produce a molten sample According to such embodiments, thesample is frozen in the detection step and the nuclear spin transitionsin the at least one spin half nucleus of the analyte in the frozensample are detected by solid-state NMR.

In another embodiment, the methods do include a step of melting thesample to produce a molten sample. According to such embodiments, thesample is molten in the detection step and the nuclear spin transitionsin the at least one spin half nucleus of the analyte in the moltensample may be detected by liquid-state NMR Alternatively, the nuclearspin transitions in the at least one spin half nucleus of the analyte inthe molten sample may be detected by MRI According to this lastembodiment, at least a portion of the molten sample that includespolarized analyte is administered (e.g., by injection, ingestion,inhalation, etc.) to a subject prior to detection. In certainembodiments (e g., when the polarizing agent is toxic) the polarizedanalyte may be separated from the polarizing agent prior toadministration. U.S Pat. No. 6,311,086 (the contents of which areincorporated herein by reference) describes several methods forachieving such a separation (e.g., physical and chemical separation orextraction techniques).

In general, the methods may be used to polarize any analyte Withoutlimitation, the analyte may be a protein or nucleic acid. In certainembodiments, the analyte may be a protein or nucleic acid that iscovalently attached to the polarizing agent. For example, in the case ofpolarizing agents defined by Formula I, R¹, R², or both R¹ and R² are ananalyte (e.g., a protein) covalently attached to the polarizing agent).

Numerous solid-state and liquid-state NMR methods have been developed tostudy the structures of these biomolecules, e.g., one dimensionaltechniques, multi-dimensional techniques, including without limitationtechniques that rely on NOESY, ROESY, TOCSY, HSQC, HMQC, etc. typepolarization transfers and combinations thereof Any of these techniquesand variants thereof may benefit from the enhanced NMR signals that canbe provided by the methods described herein . The methods may also beadvantageously used to improve the detection of analytes (e.g.,metabolites) that are present in a sample at low concentrations (e.g.,less than 1 µM, less than 100 nM, less than 10 nM or even less than 1nM). When the analyte is being used as an imaging agent for an MRIexperiment then it will preferably include at least one spin halfnucleus with a long T₁ relaxation time This will ensure that theenhancement is not lost by relaxation in between the polarizing anddetecting steps. For example, U.S. Pat. No. 6,311,086 describes imagingagents that include spin half nuclei with T₁ relaxation times of atleast 6 seconds at 310 K in D₂O in a magnetic field of 7 T. It will beappreciated that any of the imaging agents that are described in U.S.Pat. No. 6,311,086 may be used as an analyte in a method describedherein. It is also to be understood that any known MRI technique may beused to image the spatial distribution of a polarized analyte onceadministered to a subject (e.g., see MRI in Practice Ed. by Westerbrooket al., Blackwell Publishing, Oxford, UK, 2005, the contents of whichare incorporated herein by reference).

Any spin half nucleus within the analyte may be polarized according tothe methods described herein. In one embodiment, the spin half nucleusis a ¹H nucleus. In one embodiment, the spin half nucleus is a ¹³Cnucleus. In one embodiment, the spin half nucleus is a ¹⁵N nucleus. Inone embodiment, the spin half nucleus is a ¹⁹F nucleus. The spin halfnucleus may be present in the analyte at natural abundance levels.Alternatively, stronger signals may be obtained if the spin half nucleus(e.g., ¹³C, ¹⁵N, ¹⁹F, etc.) is enriched at one or more positions withinthe analyte. A variety of methods are known in the art for enriching oneor more sites of an analyte (e.g., a protein, nucleic acid, metabolite,imaging agent, etc.). When the at least one spin half nucleus has aγ-value smaller than that of ¹H (e.g., ¹³C, ¹⁵N, ¹⁹F, etc.) then incertain embodiments, the step of polarizing may further involveirradiating the frozen sample with radiation having a frequency thatcauses cross-polarization between a ¹H nucleus present in the sample(e.g., without limitation from ¹H₂O) and the at least one spin halfnucleus of the analyte.

The methods described herein may be performed under any magnetic fieldstrength. In one embodiment the field may have a strength in the rangeof about 0.1 T to about 30 T. For example, some of the experiments thatare described herein were performed at 5 T. The radiation for excitingelectron spin transitions in the unpaired electron(s) of the polarizingagent at these fields will be in the range of about 2.8 GHz to about 840GHz For examples, the radiation in the experiments that are describedherein was from a 140 GHz gyrotron.

When studying molten samples (e.g., by liquid-state NMR), the sample maybe recycled by freezing the sample, rcpolarizing the at least one spinhalf nucleus of the analyte by irradiating the frozen sample withradiation having a frequency that excites electron spin transitions inthe polarizing agent, remelting the frozen sample to produce a moltensample, and redetecting nuclear spin transitions in the at least onespin half nucleus of the analyte in the molten sample This process canbe repeated for as many cycles as needed. This can be used, e.g., tosignal average NMR signals and thereby further enhance the sensitivityof the NMR experiment. The freezing step can generally be achieved bycooling the sample until it reaches a solid state. In certainembodiments, the sample can be cooled to a temperature of less thanabout 200 K. For example, the sample may be cooled to a temperature inthe range of about 1 K to about 100 K. Some of the experiments that aredescribed herein involved cooling the sample to a temperature of about100 K. In one embodiment, the freezing step may be completed in lessthan about 2 minutes, e.g.. less than about 1 minute.

In general, once a frozen sample has been polarized, it can beoptionally melted prior to signal detection using any suitable method.In certain embodiments, this is achieved by exposing the frozen sampleto radiation having a wavelength of less than about 100 µm, e.g., in therange of about 0.5 µm and about 50 µm. In one embodiment, the radiationmay come from a laser, e.g., a CO₂ laser . In another embodiment, theradiation may come from a lamp, e.g., an infra-red lamp. The frozensample can be exposed to the radiation using an optical fiber. This willtypically involve coupling the radiation (e.g., from a laser or lamp) toone end of the fiber, e.g., using a lens. In one embodiment, the sampleis within a cylindrical rotor The rotor can be made of quartz whichallows both microwave radiation (e.g, the 140 GHz radiation from agyrotron) and infra-red radiation (eg. from a CO₂ laser) to reach thesample. In some cases, the quartz rotor does not crack when exposed tomultiple freeze-thaw cycles Finally, the use of a cylindrical rotor canenable the sample to be spun during the melting step (and optionallyduring other steps including the detecting step) which can significantlyimprove melting homogeneity and time. In some examples, samples aremelted in less than about 1 second.

By way of non-limiting illustration, examples of certain embodiments ofthe present disclosure are given below.

EXAMPLES Example 1: Peptide-Based Biradicals for Dynamic NuclearPolarization of Solid-State Nuclear Magnetic Resonance Spectroscopy

The modular and facile generation of nitroxide-based biradicalpolarizing agents for dynamic nuclear polarization (DNP) solid-state NMRvia solid-phase peptide synthesis is described. Four representativepeptides were prepared to illustrate the concept, each containing twoTOAC spin label amino acids(2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid) at positions iand i+1 or i and i+2, including TOAC-TOAC, acetyl-TOAC- TOAC,TOAC-TOAC-Ser and TOAC-Ser-TOAC. Electron-electron dipolar couplingscomputed from geometries optimized using broken-symmetry DFT are foundto be large, in the ~50-60 MHz range. To assess their utility forpotential biomolecular DNP solid-state NMR applications, the fourpeptide-based biradicals were used as exogenous polarizing agents toenhance ¹H-¹³C CP-MAS solid-state NMR spectra of ¹³C,¹⁵N-proline in theusual 6:3:1 d₈-glycerol/D₂O/H₂O matrix at 600 MHz ¹H frequency andtemperature of ~100 K. At their optimal concentrations (~10-15 mM), thepeptide-based biradicals yielded absolute DNP NMR signal enhancementsranging between ~10 and ~20 and absolute enhancements per unit timeranging between ~5 and ~10, with TOAC-TOAC-Ser providing the highestsignal enhancement factors. These results compare favorably with thoseobtained for the well-established TOTAPOL and AMUPol polarizing agents,which under the same conditions yielded absolute NMR signal enhancementsof ~6 and ~30, respectively, and absolute enhancements per unit time of~3 and ~18, respectively. The fact that such peptide-based biradicalpolarizing agents displaying an array of physicochemical propertiestailored toward different applications, including the possibility totarget specific sites in biomolecules by covalent attachment ornon-covalent binding, can be readily synthesized makes them a promisingtool for DNP solid-state NMR.

Introduction

Dynamic nuclear polarization (DNP) is a powerful technique in increasingthe overall sensitivity of solid-state nuclear magnetic resonance(ssNMR) spectroscopy. DNP employs a microwave-driven transfer ofpolarization from free electrons to surrounding nuclei, to achievehyperpolarization in a given magnetic field. While this method has beenknown since the 1950s, recent technologies have enabled DNP to becomecoupled to high-field ssNMR with the development of gyrotrons capable ofgenerating high-powered, high-frequency microwaves. In theory, themaximum signal enhancement that may be achieved using DNP is a ratio ofthe electron to proton Larmor frequencies, γ_(e)/ γ_(H)~660. Currently,DNP signal enhancements of 30 to 50 have been achieved for biologicalsamples and over 200 in small molecules and materials.

A number of methods have been paired with DNP to optimize the techniquefor studying small molecules, biological systems and syntheticmaterials. Low temperatures are employed to maximize the DNP effect byexploiting longer spin relaxation times at low temperatures . Smallbiradical molecules have been synthesized to serve as an exogenousparamagnetic source and to utilize the cross-effect (CE) mechanism ofDNP Glass-forming solvents, such as glycerol and tetrachloroethane(TCE), are used to dissolve these biradical polarizing agents and tosuspend them throughout a sample-of- interest in a cryo-protectingglass. Magic-angle spinning (MAS) ssNMR is employed to study thesefrozen glasses, with the use of cryogenic probes, to overcome chemicalshift anisotropies found in solids and frozen solutions . Recently,additional methods such as systematic biradical design, isotopicdeuteration of polarizing agents, fast-spinning MAS, andmulti-dimensional NMR experiments have been employed to further enhancethis strategy.

Nitroxide radicals are the preferred radical in DNP because they arestable and easily incorporated into organic syntheses. Nitroxide-basedbiradicals were developed by synthetically tethering together two2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO)- moieties. By fixing theinter-electron distance through biradical design, the efficiency of theCE mechanism of DNP greatly increases, resulting in larger DNPenhancements. Common biradicals include TOTAPOL, AMUPOL and TEKPOL,which can reach DNP enhancements of over 200. Biradicals have beendesigned to shorten inter-electron distances, to restrain dinitroxideg-tensors in orthogonal orientations, to increase electron relaxationtimes (T_(1e), T_(2e)) and to increase solubilities.

In this example, a new category of nitroxides biradicals based onpeptide synthesis and the amino acid derivative TOAC is described. Solidphase peptide synthesis (SPPS) is a popular technique for synthesizingshort peptides in a quick and controlled fashion. By using TOAC as amono-nitroxide building block, biradical peptides can quickly besynthesized to different inter-nitroxide lengths and amino acidcompositions. Herein, four example TOAC-based biradical peptides havebeen prepared and evaluated: TOAC-TOAC (TT), TOAC-Ser-TOAC (TST),TOAC-TOAC-Ser (TTS) and acetyl-TOAC-TOAC (ATT). In addition, thegeometries of these biradicals were evaluated using broken symmetry DFTcalculations, their biradical characteristics were investigated bycontinuous wave (CW), X-band EPR. spectroscopy, and their DNP-ssNMRcharacteristics as polarizing agents were identified.

Materials and Methods

Biradical peptide synthesis: TOAC-based biradical peptides TOAC-TOAC(TT), TOAC-Ser-TOAC (TST), TOAC-TOAC-Ser (TTS), and acetyl-TOAC-TOAC(ATT) were synthesized via Fmoc-bascd solid-phase peptide synthesis.Briefly, Fmoc-2,2,6,6-tetramethylpipcridinc-N-oxyl-4-amino-carboxylicacid (Fmoc-TOAC-OH, Chem-IMPEX, CAS#93372-25-9) was coupled to Wangresin using HATU, HOAt, and DIPEA. dissolved in DMF. The reaction vesselwas shaken at room temperature for 24 hr. The Fmoc-protecting group wasremoved with 20% piperidine/DMF. Subsequent amino acid or TOAC-couplingswere then repeated until desired product was prepared Then, the peptidewas cleaved from the resin using 95%TFA/2.5%H₂O/ 2.5% TIS. The filtratewas concentrated under vacuum and re-dissolved in methanol. The crudeproduct was purified by reverse-phase HPLC (C₁₈, 50 mM NH₄CH₃COO pH=5.0,60% CH₃CN/H₂O) and lyophilized. Nitroxide-radicals are regenerated byre-dissolving product in 0.5 M NH₄CH₃COO pH 9.5 and let sit overnight at4° C. Ammonium acetate is removed via HPLC (C₁₈, H₂O, 60% CH₃CN/H₂O).Pure product is lyophilized and stored at 4° C.

Detailed synthetic protocols for TT, TST, TTS, and TTS are describedbelow.

EPR Spectroscopy: All spectra were obtained on a Bruker EMXPlus EPRspectrometer (CW. X-band) at room temperature. A standard concentrationcurve was made using 4-hydroxyl-2,2,6,6-tetramethyl-1-oxyl (TEMPOL,Sigma-Alrich, CAS#2226-96-2) dissolved in 3:2 glycerol/H₂O and packedinto a glass capillary. TEMPOL concentrations were confirmed andstandardized by UV-Vis spectroscopy Biradical EPR-profiles are obtainedby dissolving activated TOAC-based biradical peptides in methanol atconcentrations of approximately 5 mM to 20 mM Biradical concentrationsof DNP samples in 6:3:1 d8-glycerol/D₂O/H₂O are determined using theTEMPOL standard curve.

DNP-SSNMR Sample Preparation: AMUPOL was obtained from SATT SUD-EST(Marseille, France) TOTAPOL was purchased from DyNuPol, Inc. (Newton,MA). These nitroxides were used as provided by the manufacturer withoutany further purification. AMUPOL and TOAC-TOAC-Ser were dissolved in1.25 M 13C,15N-L-proline 3:1 D₂O/H₂O solution. d8-glycerol was added byweight to a final ratio of 6:3:1 d8-glycerol/D₂O/H₂O and a final prolineconcentration of 0.5 M. Lyophilized TOAC-TOAC and Acetyl-TOAC-TOAC weredissolved in 1.25 M 13C,15N-L-proline, 1 eq. ofNaOH, 3:1 D₂O/H₂Osolution d8-glycerol was added by weight to a final ratio of 6:3:1d8-glycerol/D₂O/H₂O and a final proline concentration of 0.5 M. TOTAPOLwas dissolved directly into a solution of 0.5 M 13C,15N-L-proline in6:3.1 d8-glycerol/D₂O/H₂O. Biradical concentrations were measured byEPR. Samples were flash frozen and thawed 10 times each. Finally, 23 µLof each solution was pipetted directly into a clean, dry 3.2 mm sapphirerotor and equipped with a silicone plug.

DNP-SSSrMR Spectroscopy: All experiments were performed on a BrukerAvance III HD Wide-Bore 14.1 T spectrometer equipped with a 7.2 Tgyrotron cryogenic magnet and a 3.2 mm, triple-resonance (HXY),cryogenic LT-MAS probe. Samples were packed into Bruker 3.2 mm sapphirerotors, each with a silicone plug and a zirconium cap. Proline sampleswere spun at a magic angle frequency of 8000 Hz, with temperaturesranging from 97 K to 107 K. Microwave field power curves were performedfor each biradical, to determine the optimal applied MW power for DNPenhancement. Microwaves are set to 145 mA (0.34 V) for AMUPOL, 100 mA(0.10 V) for TOTAPOL, and 115 mM (0.15 V) for TOAC-TOAC DNP build-uptimes were measured from a saturation-recovery experiment ¹H T₁relaxation times were measured from two inversion-recovery experiments,one with the microwaves turned on and one with the microwaves turnedoff. 1D ¹H-¹³C cross-polarization experiments were recorded of eachsample. For 10 min experiments, recycle delays were set to 1.256 x τDNPand number of scans are adjusted to set total experiment time toapproximately 10 minutes. Each sample used 4 dummy scans.Signal-to-noise ratios are determined using TOPSPIN 3.5 and are dividedby total experiment times (in seconds). Each sample was made from thesame proline stock solution, so it was assumed that prolineconcentrations were identical.

DFT Calculations: Electronic structure calculations were performed usingbroken-symmetry density functional theory (DFT) at a ωB97X-D/6-31+G*level. C-PCM solvation model with a dielectric constant of 59.3 is alsoapplied. A dielectric constant of 59.3 matches that of 3:2 glycerol/H₂O.Calculations of bTUrea and TOTAPOL are used as standards. 1-5 localminimum energy conformations are found in the gas phase for TT, TST, TTS& ATT and the solvent model is applied to each of these. Nitroxidedistances and relative orientations are extracted from these structuresand weighted averages are determined based on their relative abundancesat 100 K.

Results and Discussion

The unnatural amino acid 2,2,6,6-tetramethyl-1-oxyl-4-amino-4-carboxylicacid (TOAC) contains a stable nitroxide radical and has been used as anESR spin label. An Fmoc-protecting group can be added for use inFmoc-based solid phase peptide synthesis (SPPS) This allows TOAC to beincorporated into a peptide at a specific location to serve as a spinprobe. By creating TOAC-based biradical peptides though SPPS, theinter-electron distances, solubilities and geometries of each polarizingagent can be customized by inserting additional amino acids or byfunctionalizing the C- and/or N-terminus of the peptide (e.g.,acetylating the N-terminus).

The established biradicals AMUPOL and TOTAPOL are used as standardpolarizing agents in aqueous media TOTAPOL contains two TEMPO-moietieslinked by a 5-atom tether. AMUPOL is the leading biradical for use inaqueous media as the nitroxide flanking methyl groups found in TEMPO,are replaced by spirocyclohexyl groups (FIG. 1 ). This results in longerT_(2e) relaxation times and increased DNP enhancements. Additionally,AMUPOL has an increased solubility in glycerol-water mixtures thanTOTAPOL, and other nitroxide biradicals. This higher solubility enablesAMUPOL to form a good glass in glycerol-water mixtures and to becompatible with biological systems

Four TOAC-based biradical peptides were synthesized by SPPS usingFmoc-TOAC-OH, as described in Scheme 1. TOAC-TOAC (TT) is a short,biradical dipeptide; TOAC-Ser-TOAC contains a spacer amino acid;TOAC-TOAC-Ser (TTS) contains an terminal serine; and acetyl-TOAC-TOAC(ATT) contains an acetylated amide (FIG. 1 ). TOAC residues were coupledto a solid p-benzyloxybenzyl alcohol resin (Wang resin) using HATU/HOAtcoupling reagents in the presence of DIPEA. Following SPPS, nitroxideradicals needed to be regenerated as they were oxidized in the strongacidic conditions required for cleavage. This was accomplished bytreatment in 10-molar excess ammonium acetate (H₄CH₃COO) with a pHadjusted to 9.5 for 24 hours. NH₄CH₃COO was then removed through abuffer exchange via HPLC.

Scheme 1. Synthetic pathway of TOAC-TOAC by solid-phase peptidesynthesis using Wang resin andFmoc-2,2,6,6-ietramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid(Fmoc-TOAC-OH).

Broken symmetry DFT. ωB97X-D/6-31 +G calculations were performed toexamine the geometries of the TOAC-based biradical peptides. Brokensymmetry DFT was used to break the alpha and beta spin symmetry in orderto properly capture the open-shell singlet character Up to five of thelowest energy conformers were found in the gas phase for each biradical.These geometries were then optimized using a C-PCM solvation model witha dielectric constant of 59.3 was also applied to mimic DNP solventconditions of 60% glycerol Inter-nitroxide distances were extracted asthe average between the N-N distance and the O-O distance. Theelectron-electron dipolar coupling constant, D_(ee), was calculated as afunction of the inter-nitroxide distance. Angles between C₂NO planes andNO-vectors were also measured. Weighted averages of these propertieswere calculated using each conformers’ abundance at 100 K, using theirrelative energies (Table 1)

TABLE 1 Internuclear distances and geometries of TOAC-based biradicalpeptides N-N distance (Å) O-O distance (Å) Dipolar coupling constant,D_(ee) (MHz) Angle between C₂NO planes, θ Angle between NO vectors, φATT 8.747 10.96 59.7 34.8 128.9 TST 9.171 10.94 56.4 48.1 89.6 TT 9.16311.61 50.5 28.9 163.6 TTS 9.240 11.70 49.3 35.0 160.2

TOAC directly bound to TOAC only has a 2-atom linkage betweenTEMPO-moieties, shorter than any previous nitroxide biradical. Thisresults in short inter-nitroxide distances and, subsequently, largedipolar coupling constants. ATT has the shortest inter-electron distanceof 9 265 Å and a dipolar coupling constant of 59.7 MHz In comparison,TOTAPOL, has an inter-electron distance of 13.1 Å and D_(ee) of 23.2MHz, and AMUPOL, has an inter-electron distance of 11.61 Å and a D_(ee)of 35.4 MHz. These TOAC-TOAC peptides (TT, TTS, ATT) share similargeometries between inter-electron distance and angles betweenTEMPO-groups. Also, the short linkage between TOAC residues leads a morerestricted geometry. Interestingly, TST has the second shortestinter-nitroxide distance despite having an additional amino acidseparating the TEMPO-moieties. This is a result of a hydrogen bondbetween terminal amine and carboxylic groups, which pulls the TOACresidues together. But the longer backbone will likely result in alarger sampling of orientations with less favorable DNP characteristics.

Room temperature, continuous wave (CW), X-band EPR spectra werecollected for each biradical peptide These spectra all display asignature five-line pattern 3-lines coincide with a monomeric nitroxideradial due to electron-nitrogen (¹⁴N) hyperfine coupling (A_(N)) whilean additional two are present due to coupled electrons Biradicalspossessing these five-peak spectra can be regarded as having an averageexchange integral (J) larger than the A_(N). Biradicals possessing only3-peaks are assumed to have J ≈ A_(N) (45 MHz) The EPR spectra changesbetween solvents (methanol, 60% glycerol) because the rate of molecularreorientation depends on the solvent viscosity. Anisotropies present inthe g-tensor and ¹⁴N hyperfine tensor results in increased linewidths inviscous solvents. EPR spectra of biradicals dissolved in 60% glycerolare used for determining quantitative electron spin concentrationsMonoradical TEMPOL dissolved in 60% glycerol is used as a standard, withconcentrations confirmed by UV-Vis spectroscopy. EPR spectra arecollected with a 150 G sweep width, microwave power of 2.000 mW (belowthe saturation condition). 7.0 s sampling time and 1500 points. Thedouble integration of each sample was recorded and compared to theTEMPOL concentration curve to determine the electron spin concentration.Using this method, a series of samples with increasing biradicalconcentration are prepared for each TOAC-based biradical peptide,AMUPOL, and TOTAPOL. Between four and six samples are prepared for eachbiradical, with final dinitroxide concentrations ranging from 5 mM to 30mM. Each sample was prepared and packed in identical fashion, using thesame stock solutions and d8-glycerol to ensure that a final prolineconcentration of 0.5 M was consistent among each sample This allowed forthe direct comparison of the DNP enhancement and depolarizing effect ofeach biradical.

¹H-¹³C cross-polarization experiments are recorded for each 0.5 M¹³C,¹⁵N-L-proline sample with varying concentrations of each biradical.DNP enhanced spectra are collected in the presence of microwaveirradiation, set at a radical-dependent power level. Spectra in theabsence of DNP enhancement are recorded with the microwave irradiationturned off. Microwave-off spectra are collected at a temperature of 97 Kand the temperature of microwave-on spectra vary from 101 K to 107 K.These temperature differences are not accounted for when directlycomparing the on-to-off signal enhancement, commonly known as the DNPenhancement, ε_(DNP) (equation 1).

$\text{ε}_{DNP} = \frac{intensity( {MW\mspace{6mu} on} )}{intensity( {MW\mspace{6mu} off} )} - 1$

Absolute enhancement, ε_(Abs), is determined through the signalintensity difference between a biradical-doped sample and an undopedsample of identical proline concentration (equation 2)

$\text{ε}_{Abs} = \frac{intensity( {MW\mspace{6mu} on} )}{intensity( {MW\mspace{6mu} off} )} - 1$

ε_(Abs) values are always smaller than ε_(DNP) values because thepresence of biradical depolarizes nearby nuclei. The amount ofdepolarization varies with the nature of each biradical, the spinconcentration and the MAS frequency. The enhancement profiles of TT,TST, TTS and ATT are shown in FIGS. 2A-2D. 15 mM ATT is found to havethe greatest ε_(DNP) of 48 while 6 mM TTS has the largest ε_(Abs) of 21.TT and TST have more modest enhancements (ε_(DNP) = 25, ε_(Abs) =11-12). These biradical peptides outperform TOTAPOL in these sameconditions (ε_(DNP) = 21. ε_(Abs) = 9) but fall behind AMUPOL (ε_(DNP) =107, ε_(Abs) = 37). It should be noted that maximum ε_(DNP) and ε_(Abs)values do not occur at the same biradical concentration

Another important factor to determine the optimal sample composition isthe DNP buildup time, τ_(DNP), which is the amount of time required forthe enhanced NMR signal to recover under DNP conditions. Lowtemperatures slow down spin relaxation to where the ¹H T₁ relaxationtimes of proline, in the absence of any radical, is on the order of 60 sat 100 K. This increases the required delay time between scans and makessignal averaging tedious. The addition of paramagnetic speciesreintroduces spin relaxation as a function of radical concentration.τ_(DNP) is a useful measure of ¹H T₁ relaxation and DNP propagationthroughout a sample The optimal recycle delay between scans can then beset to 1.26 × τ_(DNP). DNP build up times decrease with increasing TT,TST, TTS and ATT concentrations (FIGS. 2A-2D).

Neither the ε_(D) or ε_(Abs) properly measures the overall signal gainsfrom DNP in real time. A more accurate measure is a time-adjustedabsolute enhancement, ε_(Abs-time), which predicts the absolute NMRsignal enhancement possible with various biradicals by taking intoaccount τ_(DNP) times and how quickly scans can be recycled (equation3).

$\text{ε}_{Abs - time} = \frac{\varepsilon_{Abs}}{\sqrt{1.256 \times \tau_{DNP}}} - 1$

Time-adjusted absolute enhancement profiles for all six biradicalstudied here are calculated as a function of biradical concentration(FIG. 3 ) TOTAPOL has the lowest ε_(Abs-time) values as small ε_(Abs)are combined with longer τ_(DNP) times, for a given spin concentration.TST has only slightly better ε_(Abs-time) values, obtaining a maximum of4.8 at 14 mM TST. This is likely because TST and TOTAPOL, are similar insize and flexibility. TST has a larger ε_(DNP) from a large D_(ee) inits lowest energy conformation, but has a long τ_(DNP) time, relative toother biradicals.

AMUPOL has the largest ε_(DNP) but its ε_(Abs) is a factor of 3 smaller,due to large depolarizing effects. AMUPOL is still the best polarizingagent with an ε_(Abs-time) of 18.0 s^(½), but the class of TOAC-TOACbiradical peptides are not far behind. TT has the most modest ε_(DNP) of25 but exhibits the least depolarization effects, dropping only by afactor of two with an ε_(Abs) of 12. Additionally, TT exhibitssignificantly faster DNP build up times At similar concentrations of 12mM biradical, AMUPOL has a τ_(DNP) of 2.35 s while TT has a τ_(DNP) of1.60 s. These phenomena in TT are likely due to a more even distributionof biradical throughout the sample and better glass formation. ATT hasthe largest ε_(DNP) of the biradical peptides at 48 but suffers fromlarge depolarizing effects, with an ε_(Abs) of only 13. TTS appears tobe the best balance among the newly synthesized biradical peptides. TTShas an ε_(DNP) of 40 and mild depolarizing effects of a factor of two,with an ε_(Abs) of 19. While τ_(DNP) times are not as short as TT, TTShas an εAbs-time of 10.3 s^(½) due to a higher solubility and betterglass formation. Optimal biradical concentrations are determined fromthese εAbs-time profiles and these DNP properties are summarized inTable 2.

TABLE 2 DNP properties of each polarizing agent at optimal biradicalconcentrations AMUPOL TT TST TTS ATT TOTAPOL opt biradical conc. mM 12.19.33 14.4 12.7 14.5 15.5 τDNP (s) 2.35 2.07 3.90 2.59 2.58 2.45 εDNP107.4 24.6 25.3 40. 2 47.7 16.5 εAbs 30.9 11.7 10.6 18.5 13.0 5.6$\frac{\text{ε}_{Abs}}{\sqrt{1.256 \times \tau_{DNP}}}( S^{\frac{- 1}{2}} )$18.0 7.3 4.8 10.3 7.2 3.2

10-minute “real time” experiments were set up to visualize thetime-adjusted absolute enhancement (FIG. 4 ). Recycle delays were set to1.26 × τDNP and the number of scans were adjusted until a total NMRexperiment time was approximately 10 minutes. The AMUPOL signal isproportionally set to match the calculated εAbs-time . Relativeintensities of the remaining biradicals closely match the εAbs-timetrend; TT = 10.0, TST = 4.9, TTS = 12.1, ATT = 9.0, and TOTAPOL = 3.6(with respect to AMUPOL = 18.0.

The four TOAC-containing biradical peptides studied here demonstrate theability to use SPPS to generate dinitroxide polarizing agents forDNP-ssNMR spectroscopy. These four peptides have various physiochemicalcharacteristics which are tuned by pairing a serine amino acid withTOAC, changing the sequential order of residues, or the acetylation ofthe N-terminus. This results in large D_(ee) ranging from 49 to 60 MHz,increased solubility of serine containing-peptides, and overall superiorDNP utility than TOTAPOL. These biradical peptides also appear to resultin better glass formation and a more homogenous distribution throughoutthe glass, as evidenced by shorter τ_(DNP) at equal nitroxideconcentrations of both AMUPol and TOTAPOL. This is explicitly seen intwo human prion protein (huPrP23-144) fibril samples prepared withAMUPol and TTS, at identical biradical concentrations. The samplecontaining 12 mM TTS has a τ_(DNP) half that of the sample with 12 mMAMUPol, 2 s to 4 s, which allows for twice the scans in a givenexperimental time. A logical extension of this study would be thesynthesis of additional dinitroxide peptides with sequences andcompositions adjusted for desired properties. This would includepeptides containing specific amino acid sequences to non-covalentlytarget biomolecules of interest, adding hydrophobic or hydrophilicresides to tune solubilities in aqueous or organic solvents, andcovalently binding biradicals to biomolecules through peptide ordisulfide bonds. The customizable properties of this new class ofnitroxide biradical polarizing agents the potential to expand thetechniques used to study numerous systems by DNP-ssNMR

Experimental Procedures

Protocol for the Synthesis of TOAC-TOAC (TT): In a 10 mL shaker vessel,202.14 mg of Wang resin was swollen in DCM for 30 mins, then drained anddried under vacuum. Separately, 228.53 mg of Fmoc-TOAC-OH. 117.98 mg ofHOAt and 328.7.3 mg of HATU were dissolved in 3 mL of DMF Next, 299.3 µLof DIPEA was added, and the solution was mixed for 5 min. Finally, thecoupling solution was added to the 10 mL vessel, the vessel was flushedwith N₂ gas and shaken for 24 hours at RT. The solvent was drained, theresin was washed with DMF (3x) and DCM (3x), then dried under vacuum. AKaiser test was used to confirm complete coupling The resin was cappedwith 32.3 µL acetic anhydride and 27.7 µL pyridine for 30 mins. Thesolvent was drained, the resin was washed with DMF (3x) and DCM (3x),then dried under vacuum. The resin was then deprotected with 20%piperidine/DMF for 20 min (2x), rinsed with DMF (3x) and DCM (5x), anddried

The Fmoc-TOAC-OH coupling was then repeated (as described above) andshaken for 48 hours at RT. The Kaiser test was used to confirm completecoupling. The resin was then deprotected with 20% piperidine/DMF (2x),rinsed with DMF (3x) and DCM (5x), and dried. The product, TOAC-TOAC,was cleaved from the resin with 95% TFA, 2.5% TIS and 2.5% H₂O. The TFAwas collected and concentrated, then the product was precipitated byadding diethyl ether. The product was collected via centrifugation. Theproduct was redissolved in MeOH, purified by reverse-phase HPLC (C18, 50mM NH₄CH₃-COO, pH = 5.0 / CH₃CN) and lyophilized.

The TOAC-TOAC was dissolved to a concentration of 50 mM in a solution of500 mM NH₄CH₃COO pH = 9.5. The solution was vortexed to fully dissolveand gently agitated for 24 hours at RT to fully activate Buffer exchangewas performed using reverse-phase HPLC (C18, H₂O / CH₃CN), and theresulting product was lyophilized.

TT was fully characterized by Electron Spray Ionization (ESI) MassSpectrometry and Electron Paramagnetic Resonance (EPR) Spectroscopy FIG.5A shows positive mode ESI-MS of oxidized TOAC-TOAC, TT(OH). Expectedmass: 414.28 m/z Found masses: 414.27 m/z, 415.27 m/z, 416.28 m/z 95.6%TT purity. FIG. 5B shows negative and positive mode ESI-MS of activated,reduced TOAC-TOAC, TT. Expected mass: 412.27 m/z. Found masses: 411.24m/z, 412.25 m/z, 413.25 m/z, 413.26 m/z, 414.27 m/z. 90.4% TT purity.FIG. 5C shows continuous wave, X-band EPR spectrum of activatedTOAC-TOAC (500 mM NH₄CH₃COO pH 9.5, 50 mM TT) diluted to 5 mM TT inmethanol. Microwave power = 2.0 W, sampling time = 7.0 s, time constant= 1.28 s, 4 scans. FIG. 5D shows continuous wave, X-band EPR spectrum ofpurified TOAC-TOAC by reverse-phase HPLC (C18, H₂O / CH₃CN) andlyophilization . Dissolved in methanol Microwave power =2.0 W, samplingtime = 7.0 s, time constant = 1.28 s, 4 scans.

Prolocol for the Synthesis of TOAC-Ser-TOAC (TST): In a 10 mL shakervessel, 207.42 mg of Wang resin was swollen in DCM for 30 mins, thendrained and dried under vacuum. Separately, 233.08 mg of Fmoc-TOAC-OH,121.33 mg of HOAt and 336.56 mg of HATU were dissolved in 3 mL of DMF.Next, 296.3 µL of DIPEA were added and the solution was mixed for 5 minFinally, the coupling solution was added to the 10 mL vessel, the vesselwas flushed with N₂ gas and shaken for 24 hours at RT The solvent wasdrained, wash the resin with DMF (3X) and DCM (3X), then dry undervacuum. Use Kaiser test to confirm complete coupling. The resin wascapped with 32.1 µL acetic anhydride and 27.4 µL pyridine for 30 mins.The solvent was drained, the resin was washed with DMF (3x) and DCM(3x), then dried under vacuum. The resin was deprotected with 20%piperidine/DMF for 20 min (2x), rinsed with DMF (3x) and DCM (5x), anddried.

325.16 mg of Fmoc-Ser-(tBu)OHand 337.57 mg of HATU were dissolved in 3mL of DMF. Next, 296.3 µL of DIPEA was added and the solution was mixedfor 5 min. Finally, the coupling solution was added to the 10 mL vesselThe vessel was flushed with N₂ gas and shaken for 21 hours at RT. Thesolvent was drained, and the resin was washed with DMF (3X) and DCM(3X), then dried under vacuum. A Kaiser test was used to confirmcomplete coupling. The resin was deprotected with 20% piperidine/DMF(2x), rinsed with DMF (3x) and DCM (5x) and dried.

The Fmoc-TOAC-OH coupling was then performed (as described above) andshaken for 48 hours at RT. A Kaiser test was used to confirm completecoupling . The resin was deprotected with 20% piperidine/DMF (2x),rinsed with DMF (3x) and DCM (5x) and dried. The product, TOAC-Ser-TOAC,was cleaved from the resin with 95% TFA, 2.5% TIS and 2.5% H₂O. Theproduct was collected and concentrated the TFA, then precipitated byadding diethyl ether The product was collected via centrifugation. Theproduct was then redissolved in MeOH, purified by reverse-phase HPLC(C18, 50 mM NH₄CH₃COO pH = 5.0 / CH₃CN) and lyophilized.

TST was dissolved to a concentration of 50 mM in a solution of 500 mMNH₄CH₃COO pH = 9.5. The solution was vortexed to fully dissolve the TTS,and the solution was gently agitated for 24 hours to fully activate .Buffer exchange was performed using reverse-phase HPLC (C18, H₂O /CH₃CN) and the resulting product was lyophilized.

TST was fully characterized by Electron Spray Ionization (ESI) MassSpectrometry and Electron Paramagnetic Resonance (EIIR) Spectroscopy.FIG. 6A shows negative mode ESI-MS of reduced TOAC-Ser-TOAC, TST.Expected mass 499.30 m/z. Found masses: 498.3 m/z. 499.3 m/z, 500.3 m/z.91.3% TST purity. FIG. 6B shows continuous wave, X-band EPR spectrum ofactivated TOAC-Ser-TOAC (500 mM NH₄CH₃COO pH 9.5, 50 mM TST) diluted to5 mM TST in methanol. Microwave power = 2.0 W, sampling time =7.0 s,time constant = 1.28 s, 128 scans.

Protocol for the Synthesis of TOAC-TOAC-Ser (TTS): In a 10 mL shakervessel, 333.6 mg of Fmoc-Ser(tBu)-Wang resin (0.51 meq/g) was swollen inDCM for 30 mins, then drained and dried under vacuum. Separately. 225.98mg of Fmoc-TOAC-OH, 115.46 mg of HOAt and 325.15 mg of HATU weredissolved in 3 mL of DMF Next, 296.3 µL of DIPEA was added and thesolution was mixed for 5 min. Finally, the coupling solution was addedto the 10 mL vessel, the vessel was flushed with N₂ gas and shaken for24 hours at RT. The solvent was drained, and the resin was washed withDMF (3X) and DCM (3X), then dried under vacuum. A Kaiser test was usedto confirm complete coupling The resin was capped with 35.8 µL aceticanhydride and 30.5 µL pyridine for 30 mins. The solvent was drained, andthe resin was washed with DMF (3x) and DCM (3x), then dried undervacuum. The resin was deprotected with 20% piperidine/DMF for 20 min(2x), rinsed with DMF (3x) and DCM (5x) and dried

The Fmoc-TOAC-OH coupling was repeated (as described above) and shakenfor 48 hours at RT A Kaiser test was used to confirm complete coupling.The resin was deprotected with 20% piperidine/DMF (2x), rinsed with DMF(3x) and DCM (5x) and dried. The product, TOAC-TOAC-Ser, was cleavedfrom the resin with 95% TFA, 25% TIS and 2.5% H₂O. The product wascollected and the TFA was concentrated, then the product wasprecipitated by adding diethyl ether. The product was collected viacentrifugation. The product was redissolved in MeOH, purified byreverse-phase HPLC (C18, 50 mM NH₄CH₃COO pH=5.0/CH₃CN) and lyophilized.

TOAC-TOAC-Ser was dissolved to a concentration of 50 mM in a solution of500 mM NH_(4C)H3COO pH = 9.5. The solution was vortexed to fullydissolve the TTS, and gently agitated for 24 hours to fully activateBuffer exchange was performed using reverse-phase HPLC (C18, H₂O/CH₃CN)and the resulting product was lyophilized.

TTS was fully characterized by Electron Spray Ionization (ESI) MassSpectrometry and Electron Paramagnetic Resonance (EPR) Spectroscopy.FIG. 7A shows negative mode ESI-MS of reduced TOAC-TOAC-Ser, TTSExpected mass: 499.30 m/z Found masses: 498.3 m/z, 499.3 m/z, 500.3 m/z.91.3% TST purity. FIG. 7B shows continuous wave, X-band EPR spectrum ofactivated TOAC-TOAC-Ser (500 mM NH₄CH₃COO pH 9.5, 50 mM TST) diluted to5 mM TTS in methanol. Microwave power = 2.0 W, sampling time = 7.0 s,time constant = 1.28 s, 128 scans.

for the Synthesis of Acetyl-TOAC-TOAC (ATT): In a 10 mL shaker vessel.207.76 mg of Wang resin was swollen in DCM for 30 mins, then drain anddry under vacuum. Separately, 228.69 mg of Fmoc-TOAC-OH, 118.30 mg ofHOAt and 330.49 mg of HATU were dissolved in 3 mL of DMF. Next, 302.8 µLof DIPEA was added, and the solution was mixed for 5 min. Finally, thecoupling solution was added to the 10 mL vessel, the vessel was flushedwith N₂ gas and shaken for 24 hours at RT. The solvent was drained, theresin was washed with DMF (3X) and DCM (3X), then dried under vacuum. AKaiser test was used to confirm complete coupling . The resin was cappedwith 35.8 µL acetic anhydride and 30.5 µL pyridine for 30 mins Thesolvent was drained, and the resin was washed with DMF (3x) and DCM(3x), then dried under vacuum. The resin was deprotected with 20%piperidine/DMF for 20 min (2x), rinsed with DMF (3x) and DCM (5x) anddried.

The Fmoc-TOAC-OH coupling was then repeated (as described above) andshaken for 48 hours at RT. A Kaiser test was used to confirm completecoupling. The resin was deprotected with 20% piperidine/DMF (2x), rinsedwith DMF (3x) and DCM (5x) and dried

Next, 32.8 µL acetic anhydride and 485 µL triethylamine in 3 mL DMF wereadded to the shaker vessel, and the solution was shaken for 4 hours atRT. This procedure was repeated and a Kaiser test was then used toconfirm complete coupling. The resin was then rinsed with DMF (3x) andDCM (5x) and dried.

The product, acetyl-TOAC-TOAC, was cleaved from the resin with 95% TFA,2.5% TIS and 2.5% H₂O. The product was collected and the TFA wasconcentrated, then the product was precipitated by adding diethyl ether.The product was collected via centrifugation Then, the product wasredissolved in MeOH, purified by reverse-phase HPLC (C18, 50 mMNH₄CH₃COO pH = 5.0/CH₃CN) and lyophilized.

The product was dissolved to a concentration of 50 mM in a solution of500 mM NH₄CH₃COO pH = 9.5. The solution was vortexed to fully dissolvethe product and then gently agitate for 24 hours to fully activate.Buffer exchange was performed using reverse-phase HPLC (C18, H₂O /CH₃CN) and the resulting product was lyophilized.

ATT was fully characterized by Electron Spray Ionization (ESI) MassSpectrometry and Electron Paramagnetic Resonance (EPR) Spectroscopy.FIG. 8A shows negative mode ESI-MS of reduced Acetyl-TOAC-TOAC, ATT.Expected mass: 454.28 m/z. Found masses: 453.3 m/z, 454.3 m/z, 455.3m/z. 907.5 m/z, 908.5 m/z. 95.1% ATT purity. FIG. 8B shows continuouswave, X-band EPR spectrum of activated Acetyl-TOAC-TOAC in methanol.Microwave power = 2.0 W, sampling time = 7.0 s, time constant = 1.28 s,128 scans

Preparation of¹³C,¹⁵N-L-Proline Samples for DPN-SSNMR: AMUPOL wasobtained from SATT SUD-EST (Marseille, France). TOTAPOL was purchasedfrom DyNuPol, Inc. (Newton, MA). These nitroxides were used as providedby the manufacturer without any further purification. TOAC-basedbiradical peptides (TT, TST, TTS, ATT) were purified by HPLC,lyophilized, and used as dry powder.

AMUPOL and TTS were dissolved in 1.25 M ¹³C,¹⁵N-L-proline 3:1 D₂O/H₂Osolution d8-glycerol was added by weight to a final ratio of 6:3:1d8-glycerol/D₂O/H₂O and a final proline concentration of 0.5 M. Dry TTand ATT were dissolved in 1.25 M 13C,15N-L-proline, 1 molar eq. of NaOH,3:1 D₂O/H₂O solution. d8-glycerol was added by weight to a final ratioof 6:3:1 d8-glycerol/D₂O/H₂O and a final proline concentration of 0.5 MTOTAPOL was dissolved directly into a solution of 0.5 M13C,15N-L-proline in 6:3:1d8-glycerol/D₂O/H₂O.

For EPR spectroscopy, 25 µL of each solution are loaded into a sealedglass capillary using a Hamilton syringe. Biradical concentrations werethen determined by EPR. Samples were flash frozen and thawed 10 timeseach. Finally, 23 µL of each solution was pipetted directly into aclean, dry 3.2 mm sapphire rotor and equipped with a silicone plug.

FIG. 9 shows the use of EPR spectroscopy to determine nitroxideconcentration 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL,172.24 g/mol) was dissolved in 3.2 glycerol/H₂O. Concentrations wereconfirmed and standardized by UV-Vis spectroscopy (λ=240 nm). EPRspectroscopy was performed on an X-band, Bruker EMX-Plus spectrometer atroom temperature. EPR parameters: center field = 3350 G, microwave power= 2.0 W, sampling time = 7.0 s, time constant = 1.28 s, 4 scans Doubleintegration of the EPR spectrum and the Q-factor of the resonator wasused to determine the electron-spin concentration.

FIG. 10 shows the EPR spectra of biradical DNP samples. 12 mM AMUPOL, 16mM TOAC-TOAC, 15 mM TOTAPOL, 17 mM TOAC-Ser-TOAC, 17 mM TOAC-TOAC-Serand 16 mM Acetyl-TOAC-TOAC biradicals were prepared in 6:3:1d⁸-glycerol/D₂O/H₂O (v/v/v) with 0.5 M ¹³C,¹⁵N-L-proline. EPRspectroscopy was performed on a continuous wave, X-band, Bruker EMX-Plusspectrometer at room temperature. EPR parameters: center field = 3350 or3308 (different cavities) G, microwave power = 2.0 W, sampling time =7.0 s, time constant = 1.28 s, 4 scans. Signals were scaled to equalintensity for clarity.

Preparation of ¹³C,¹⁵N-wt-huPrP23-144 Samples for DNP-SSNMR:¹³C,¹⁵N-wt-huPrP23-144 was expressed and purified based on previouslydescribed procedures. Fibrils were prepared using 2% wt-sced with slowinversion at 25° C. for 16 hrs Prion fibrils were pelleted bycentrifugation at 16.1 rcf and 4° C. Excess buffer was removed and thefibril pellet were washed with 6:3:1 d8-glycerol/D₂O/H₂O Pellets wereincubated in 12 mM AMUPOL or 12 mM TTS in 6:3:1 d8-glycerol/D₂O/H₂O for48 hrs at 4° C. Prion fibrils were collected via ultracentrifugation (5× 10⁵ rcf, 4° C.) and washed again with 12 mM biradical solution.Fibrils were packed into 3.2 mm sapphire rotors using a swinging buckettabletop centrifuge (3000 rcf, 4° C.). Rotors were equipped with asilicone plug and a drive cap.

Dynamic Nuclear Polarization (DNP) Solid-State Nuclear MagneticResonance (ssNMR) Spectroscopy: All experiments were performed on aBruker Avance III HD Wide-Bore 14.1 T spectrometer equipped with a 7.2 Tgyrotron cryogenic magnet and a 3.2 mm, triple-resonance (HXY),cryogenic LT-MAS probe. Samples were packed into Bruker 3.2 mm sapphirerotors, each with a silicone plug and a zirconium cap Sapphire rotorswere spun with liquid nitrogen cooled gases to achieve ultimate lowtemperatures of 97 K Constant microwave irradiation were applied to thesample through a corrugated waveguide at a set microwave power based onthe nature of the polarizing agent.

Proline samples and prion samples were spun at magic angle frequenciesof 8 kHz and 12 kHz, respectively, at temperatures ranging from 97 K to107 K. Microwave powers were set to 0.34 V for AMUPOL, 0.10 V forTOTAPOL. 0.16 V for TT, 0.14 V for TTS, 0.12 V for TST, and 0.14 V forATT.

DNP build-up times, τDNP, were measured from a saturation-recoveryexperiment. ¹H T1 relaxation times were measured from twoinversion-recovery experiments, one with the microwaves turned on andone with the microwaves turned off. 1D 1H-13C cross-polarizationexperiments are recorded of each sample For 10 min experiments, recycledelays are set to 1.256 x τDNP and number of scans are adjusted to settotal experiment time to approximately 10 minutes.

FIG. 11A shows DNP enhancements as a function of magic angle spinning(MAS) frequency from 5 kHz to 12 kHz, where microwave-on spectra arecompared to microwave-off spectra at the same spinning speed FIG. 11Bshows DNP enhancements as a function of temperature, with microwave-onspectra are compared to microwave-off spectra of approximately the sametemperature.

FIG. 12 shows microwave power curves for TOAC-TOAC (TT), TOAC-TOAC-Ser(TTS), TOAC-Ser-TOAC (TTS), and Acetyl-TOAC-TOAC (ATT). Microwave powersare arrayed from 0.06 V (90 mA current) to 0.25 V (140 mA current). DNPenhancements from measured from 1D 1H-13C CP experiments betweenmicrowave-on spectra and a microwave-off spectrum.

The DNP-SSNMR of ¹³C, ¹⁵N-wt-huPrP23-144 fibrils with both AMUPOL andTTS. The DNP properties of ¹³C, ¹⁵N-wt-huPrP23-144 fibril samplescontaining 12 mM AMUPOL and 12 mM TTS in 6:3:1 d⁸-glycerol/D₂O/H₂O(v/v/v) are included in Table 3 below FIG. 13 shows a 1D ¹H-¹³C CPexperiment of ¹³C, ¹⁵N-wt-huPrP23-144 fibrils prepared with 12 mM AMUPOLand 12 mM TTS in 6:3:1 d8-glycerol/D₂O/H₂O (v/v/v). Both DNP enhanced(microwave on) spectra and microwave off spectra are shown using optimalrecycle delays. The number of scans was adjusted to achieve a 10-minuteexperiment

TABLE 3 DNP properties of ¹³C, ¹⁵N-wt-huPrP23-144 fibril samplescontaining 12 mM AMUPOL and 12 mM TTS in 6:3:1 d⁸-glycerol/D₂O/H₂O(v/v/v) 12 mM AMUPOL 12 mM TTS εDNP 51 18 – 25 τDNP (s) 3.30 1.21 ¹H T₁(s) 3.35 1.29 Optimal recycle delay (s) 4.15 1.52

The compounds and methods of the appended claims are not limited inscope by the specific compounds and methods described herein, which areintended as illustrations of a few aspects of the claims Any compoundsand methods that are functionally equivalent are intended to fall withinthe scope of the claims. Various modifications of the compounds andmethods in addition to those shown and described herein are intended tofall within the scope of the appended claims. Further, while onlycertain representative compounds and method steps disclosed herein arespecifically described, other combinations of the compounds and methodsteps also are intended to fall within the scope of the appended claims,even if not specifically recited. Thus, a combination of steps,elements, components, or constituents may be explicitly mentioned hereinor less, however, other combinations of steps, elements, components, andconstituents are included, even though not explicitly stated.

The term “comprising” and variations thereof as used herein is usedsynonymously with the term “including” and variations thereof and areopen, non-limiting terms. Although the terms “comprising” and“including” have been used herein to describe various embodiments, theterms “consisting essentially of” and “consisting of” can be used inplace of “comprising” and “including” to provide for more specificembodiments of the invention and are also disclosed. Other than wherenoted, all numbers expressing geometries, dimensions, and so forth usedin the specification and claims are to be understood at the very least,and not as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, to be construed in light of thenumber of significant digits and ordinary rounding approaches.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

1-24. (canceled)
 25. A method comprising steps of: providing a frozensample in a magnetic field, wherein the sample includes a polarizingagent and an analyte with at least one spin half nucleus; polarizing theat least one spin half nucleus of the analyte by irradiating the frozensample with radiation having a frequency that excites electron spintransitions in the polarizing agent; optionally melting the frozensample to produce a molten sample; and detecting nuclear spintransitions in the at least one spin half nucleus of the analyte in thefrozen or molten sample, wherein the polarizing agent comprises acompound defined by Formula I

wherein L represents a direct bond or a linking group; R¹ is selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, C₁₋₁₂ alkylcarbonyl,C₁₋₁₂ alkoxycarbonyl, C₁₋₁₂ alkylcarbamyl, di(C₁₋₁₂-alkyl)carbamyl,amino acid, poly(amino acid), and poly(alkylene oxide), each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups; R²is selected from the group consisting of hydrogen, hydroxy, —OR¹¹, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, amino, C₁₋ ₁₂ alkylamino; di(C₁₋₁₂-alkyl)amino, amino acid,poly(amino acid), and poly(alkylene oxide), each optionally substitutedwith 1, 2, 3, or 4 independently selected R^(X) groups; R³ and R⁴ areindependently selected from group consisting of C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋ ₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, each optionally substituted with 1, 2, 3, or 4 independentlyselected R^(X) groups; or R³ and R⁴, together with the carbon atom towhich they are attached, form a 3-10 membered cycloalkyl or 4-10membered heterocycloalkyl ring each optionally substituted with 1, 2, 3,or 4 independently selected R^(X) groups; R⁵ and R⁶ are independentlyselected from group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋ ₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups; orR⁵ and R⁶, together with the carbon atom to which they are attached,form a 3-10 membered cycloalkyl or 4-10 membered heterocycloalkyl ringeach optionally substituted with 1, 2, 3, or 4 independently selectedR^(X) groups; R⁷ and R⁸ are independently selected from group consistingof C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋ ₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10membered aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl,C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄alkylene, 6-10 membered aryl-C₁₋₄ alkylene, 5-10 memberedheteroaryl-C₁₋₄ alkylene, each optionally substituted with 1, 2, 3, or 4independently selected R^(X) groups; or R⁷ and R⁸, together with thecarbon atom to which they are attached, form a 3-10 membered cycloalkylor 4-10 membered heterocycloalkyl ring each optionally substituted with1, 2, 3, or 4 independently selected R^(X) groups; R⁹ and R¹⁰ areindependently selected from group consisting of C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, each optionally substituted with 1, 2, 3, or 4 independentlyselected R^(X) groups; or R⁹ and R¹⁰, together with the carbon atom towhich they are attached, form a 3-10 membered cycloalkyl or 4-10membered heterocycloalkyl ring each optionally substituted with 1, 2, 3,or 4 independently selected R^(X) groups; R¹¹ is selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl,6-10 membered aryl, 5-10 membered heteroaryl, 4-10 memberedheterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10 memberedheterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄ alkylene, 5-10membered heteroaryl-C₁₋₄ alkylene, and poly(alkylene oxide), eachoptionally substituted with 1, 2, 3, or 4 independently selected R^(X)groups; and each R^(X), when present, are each independently selectedfrom OH, NO₂, CN, halo, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₄haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, cyano-C₁₋₃ alkyl, HO-C₁₋₃alkyl, amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, thio, C₁₋₆alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆ alkylsulfonyl, carbamyl, C₁₋₆alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl, carboxy, C₁₋₆ alkylcarbonyl, C₁₋₆alkoxycarbonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆alkyl)aminosulfonylamino, aminocarbonylamino, C₁₋₆alkylaminocarbonylamino, and di(C₁₋₆ alkyl)aminocarbonylamino.
 26. Themethod of claim 25, further comprising a step of freezing a samplewithin a magnetic field to provide the frozen sample in a magneticfield.
 27. The method of claim 25 , wherein the method does not includea step of melting the sample to produce a molten sample and, in the stepof detecting, the nuclear spin transitions in the at least one spin halfnucleus of the analyte in the frozen sample are detected by solid-stateNMR.
 28. The method of claim 25, wherein the method does include a stepof melting the sample to produce a molten sample and, in the step ofdetecting, the nuclear spin transitions in the at least one spin halfnucleus of the analyte in the molten sample are detected by liquid-stateNMR.
 29. The method of claim 25, wherein the analyte is a protein ornucleic acid.
 30. (canceled)
 31. (canceled)
 32. The method of claim 25,further comprising a step of freezing a sample in a magnetic field toprovide the frozen sample in a magnetic field.
 33. The method of claim25, further comprising repeating the freezing, polarizing, melting anddetecting steps at least once.
 34. (canceled)
 35. (canceled) 36.(canceled)
 37. The method of claim 25, wherein the method does include astep of melting the sample to produce a molten sample and, in the stepof detecting, the nuclear spin transitions in the at least one spin halfnucleus of the analyte in the molten sample are detected by MRI.
 38. Themethod of claim 37, wherein the spin half nucleus of the analyte has aT₁ relaxation time of at least 6 seconds at 310 K in D₂O in a magneticfield of 7 T.
 39. The method of claim 38 further comprising a step ofadministering at least a portion of the molten sample that includes theanalyte to a subject before the step of detecting.
 40. The method ofclaim 25, wherein the at least one spin half nucleus has a γ-valuesmaller than that of ¹H and the step of polarizing further comprisesirradiating the frozen sample with radiation having a frequency thatcauses cross-polarization between a ¹H nucleus present in the sample andthe at least one spin half nucleus of the analyte.
 41. The method ofclaim 40, wherein the at least one spin half nucleus is a ¹³C nucleus, a¹⁵N nucleus, a ¹⁹F nucleus, or a ¹H nucleus. 42-45. (canceled)
 46. Themethod of claim 25, wherein in the optional step of melting, the frozensample is exposed to radiation having a wavelength of less than about100 µm, such as in the range of about 0.5 µm and about 50 µm. 47-54.(canceled)
 55. The method of claim 25, wherein the magnetic field has astrength in the range of about 0.1 T to about 30 T, such as about 5 T.56. The method of claim 25, wherein the radiation has a frequency in therange of about 2.8 GHz to about 840 GHz, such as about 140 GHz. 57.(canceled)
 58. (canceled)
 59. The method of claim 25, wherein thecompound comprises three or more radicals.
 60. (canceled)
 61. The methodof claim 25, wherein the compound is defined by Formula IA

wherein L represents a direct bond or a linking group; R¹ is selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, C₁₋₁₂ alkylcarbonyl,C₁₋₁₂ alkoxycarbonyl, C₁₋₁₂ alkylcarbamyl, di(C₁₋₁₂-alkyl)carbamyl,amino acid, poly(amino acid), and poly(alkylene oxide), each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups; R²is selected from the group consisting of hydrogen, hydroxy, —OR¹¹, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, amino, C₁₋ ₁₂ alkylamino; di(C₁₋₁₂-alkyl)amino, amino acid,poly(amino acid), and poly(alkylene oxide), each optionally substitutedwith 1, 2, 3, or 4 independently selected R^(X) groups; R¹¹ is selectedfrom the group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10 memberedheterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄ alkylene, 5-10membered heteroaryl-C₁₋₄ alkylene, and poly(alkylene oxide), eachoptionally substituted with 1, 2, 3, or 4 independently selected R^(X)groups; and each R^(X), when present, are each independently selectedfrom OH, NO₂, CN, halo, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₄haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, cyano-C₁₋₃ alkyl, HO-C₁₋₃alkyl, amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, thio, C₁₋₆alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆ alkylsulfonyl, carbamyl, C₁₋₆alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl, carboxy, C₁₋₆ alkylcarbonyl, C₁₋₆alkoxycarbonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆alkyl)aminosulfonylamino, aminocarbonylamino, C₁₋₆alkylaminocarbonylamino, and di(C₁₋₆ alkyl)aminocarbonylamino.
 62. Themethod of claim 25, wherein the compound is defined by Formula IB

wherein L represents a direct bond or a linking group; R¹ is selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, C₁₋₁₂ alkylcarbonyl,C₁₋₁₂ alkoxycarbonyl, C₁₋₁₂ alkylcarbamyl, di(C₁₋₁₂-alkyl)carbamyl,amino acid, poly(amino acid), and poly(alkylene oxide), each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups; R²is selected from the group consisting of hydrogen, hydroxy, —OR¹¹, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, amino, C₁₋ ₁₂ alkylamino; di(C₁₋₁₂-alkyl)amino, amino acid,poly(amino acid), and poly(alkylene oxide), each optionally substitutedwith 1, 2, 3, or 4 independently selected R^(X) groups; R¹¹ is selectedfrom the group consisting of C₁₋₆ alkyl, C₂-₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10 memberedheterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋ ₄ alkylene, 5-10membered heteroaryl-C₁₋₄ alkylene, and poly(alkylene oxide), eachoptionally substituted with 1, 2, 3, or 4 independently selected R^(X)groups; and each R^(X), when present, are each independently selectedfrom OH, NO₂, CN, halo, C₁₋₆ alkyl, C₂-₆ alkenyl, C₂₋₆ alkynyl, C₁₋₄haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, cyano-C₁₋₃ alkyl, HO-C₁₋₃alkyl, amino, C₁₋₆ alkylamino, di(C₁₋₆ alkyl)amino, thio, C₁₋₆alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆ alkylsulfonyl, carbamyl, C₁₋₆alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl, carboxy, C₁₋₆ alkylcarbonyl, C₁₋₆alkoxycarbonyl, C₁₋₆ alkylcarbonylamino, C₁₋₆ alkylsulfonylamino,aminosulfonyl, C₁₋₆ alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl,aminosulfonylamino, C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆alkyl)aminosulfonylamino, aminocarbonylamino, C₁₋₆alkylaminocarbonylamino, and di(C₁₋₆ alkyl)aminocarbonylamino.
 63. Themethod of claim 25, wherein the compound is defined by Formula IC

wherein L represents a direct bond or a linking group; R¹ is selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, C₂-₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl,4-10 membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10membered heterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋₄alkylene, 5-10 membered heteroaryl-C₁₋₄ alkylene, C₁₋₁₂ alkylcarbonyl,C₁₋₁₂ alkoxycarbonyl, C₁₋₁₂ alkylcarbamyl, di(C₁₋₁₂-alkyl)carbamyl,amino acid, poly(amino acid), and poly(alkylene oxide), each optionallysubstituted with 1, 2, 3, or 4 independently selected R^(X) groups; R²is selected from the group consisting of hydrogen, hydroxy, —OR¹¹, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, 6-10 membered aryl,5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, amino, C₁₋ ₁₂ alkylamino; di(C₁₋₁₂-alkyl)amino, amino acid,poly(amino acid), and poly(alkylene oxide), each optionally substitutedwith 1, 2, 3, or 4 independently selected R^(X) groups; R¹¹ is selectedfrom the group consisting of C₁₋₆ alkyl, C₂-₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 4-10membered heterocycloalkyl, C₃₋₁₀ cycloalkyl-C₁₋₄ alkylene, 4-10 memberedheterocycloalkyl-C₁₋₄ alkylene, 6-10 membered aryl-C₁₋ ₄ alkylene, 5-10membered heteroaryl-C₁₋₄ alkylene, and poly(alkylene oxide), eachoptionally substituted with 1, 2, 3, or 4 independently selected R^(X)groups; R¹² is selected from the group consisting of OH, NO₂, CN, halo,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₄ haloalkyl, C₁₋₆ alkoxy,C₁₋₆ haloalkoxy, cyano-C₁₋₃ alkyl, HO-C₁₋₃ alkyl, amino, C₁₋₆alkylamino, di(C₁₋₆ alkyl)amino, thio, C₁₋₆ alkylthio, C₁₋₆alkylsulfinyl, C₁₋₆ alkylsulfonyl, carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₆alkyl)carbamyl, carboxy, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl, C₁₋₆alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl, aminosulfonylamino,C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆ alkyl)aminosulfonylamino,aminocarbonylamino, C₁₋₆ alkylaminocarbonylamino, di(C₁₋₆alkyl)aminocarbonylamino, C₃₋₁₀ cycloalkyl, 6-10 membered aryl, 5-10membered heteroaryl, 4-10 membered heterocycloalkyl, C₃₋₁₀cycloalkyl-C₁₋₄ alkylene, 4-10 membered heterocycloalkyl-C₁₋₄ alkylene,6-10 membered aryl-C₁₋₄ alkylene, 5-10 membered heteroaryl-C₁₋₄alkylene, and poly(alkylene oxide); and each R^(X), when present, areeach independently selected from OH, NO₂, CN, halo, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₁₋₄ haloalkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,cyano-C₁₋₃ alkyl, HO-C₁₋₃ alkyl, amino, C₁₋₆ alkylamino, di(C₁₋₆alkyl)amino, thio, C₁₋₆ alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆alkylsulfonyl, carbamyl, C₁₋₆ alkylcarbamyl, di(C₁₋₆ alkyl)carbamyl,carboxy, C₁₋₆ alkylcarbonyl, C₁₋₆ alkoxycarbonyl, C₁₋₆alkylcarbonylamino, C₁₋₆ alkylsulfonylamino, aminosulfonyl, C₁₋₆alkylaminosulfonyl, di(C₁₋₆ alkyl)aminosulfonyl, aminosulfonylamino,C₁₋₆ alkylaminosulfonylamino, di(C₁₋₆ alkyl)aminosulfonylamino,aminocarbonylamino, C₁₋₆ alkylaminocarbonylamino, and di(C₁₋₆alkyl)aminocarbonylamino.
 64. The method of claim 25, wherein Lrepresents a direct bond.
 65. The method of claim 25, wherein Lcomprises one or more amino acid residues.
 66. The compound of claim 65,wherein L is defined by the structure below

wherein for each occurrence in L, R¹⁴ is H and R¹³ is selected from oneof the following

or R¹³ and R¹⁴, together with the atoms to which they are attached, forma five-membered heterocycle defined by the structure below

m is an integer selected from 1, 2, 3, 4, 5, and
 6. 67. The method ofclaim 66, wherein m is 1 or
 2. 68. The method of claim 65, wherein Lcomprises a serine residue, a threonine residue, an asparagine residue,a glutamine residue, a cysteine residue, an aspartic acid residue, aglutamic acid residue, a 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylicacid residue, or any combination thereof.
 69. The method of claim 25,wherein R¹ is H, an acetyl group, or an amino acid residue selected fromthe group consisting of a serine residue, a threonine residue, anasparagine residue, a glutamine residue, an aspartic acid residue, acysteine residue, a 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acidresidue, and a glutamic acid residue.
 70. The method of claim 25,wherein R² comprises a hydroxy group or an amino acid residue selectedfrom the group consisting of a serine residue, a threonine residue, anasparagine residue, a glutamine residue, an aspartic acid residue, acysteine residue, a 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acidresidue, and a glutamic acid residue.
 71. The method of claim 25,wherein the compound is one of the following:

.